Fish Osmorregulation

Fish Osmoregulation
Fish Osmoregulation
Fish Osmoregulation
Editors
Bernardo Baldisserotto
Universidade Federal de Santa Maria

Santa Maria, RS
Brazil
Juan Miguel Mancera

Universidad de Cdiz
Cdiz, Spain
B.G. Kapoor
Formerly Professor of Zoology

The University of Jodhpur
Jodhpur, India
Science Publishers
Enfield (NH)
Jersey
Plymouth
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Published by Science Publishers, Enfield, NH, USA
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Preface
Fish lives in environments with a wide variety of chemical characteristics

(fresh, brackish and seawater, acidic, alkaline, soft and hard waters). From
an osmoregulatory point of view, fish have developed several mechanisms
to live in these different environments. Fish osmoregulation has always
attracted considerable attention and in the last years several studies have
increased our knowledge of this physiological process.
In this book several specialists have analyzed and reviewed the new
data published regarding fish osmoregulation. The chapters present an
integrative synthesis of the different aspects of this field focusing on
osmoregulation in specific environments (chapters 5 and 9) or situations
(chapter 8), function of osmoregulatory organs (chapters 11, 12 and 14),
general mechanisms (chapter 15) and endocrine control (chapters 2, 4, 6
and 16). In addition, interactions of osmoregulatory mechanisms with the
immune system (chapter 1), diet (chapter 3) and metabolism (chapter 10)
were also reviewed. Finally, new emerging techniques to study
osmoregulation are analyzed (chapters 7 and 13). We hope that this book
will provide a solid foundation for students and researchers and act as a
guide to future perspectives in this field.
The Editors
Fish Osmoregulation
Contents
Preface
List of Contributors
1. Immune and Osmoregulatory System Interaction
Alberto Cuesta, Jos Meseguer and M. ngeles Esteban
2. The Involvement of the Thyroid Gland in
Teleost Osmoregulation
Peter H.M. Klaren, Edwin J.W. Geven and Gert Flik
3. Diet and Osmoregulation
Francesca W. Ferreira and Bernardo Baldisserotto
4. The Renin-Angiotensin Systems of Fish and
their Roles in Osmoregulation
J. Anne Brown and Neil Hazon
v
ix
1
35
67
85
5. Effect of Water pH and Hardness on Survival

and Growth of Freshwater Teleosts
Jorge Erick Garcia Parra and Bernardo Baldisserotto
135
6. Arginine Vasotocin and Isotocin: Towards

their Role in Fish Osmoregulation
Ewa Kulczykowska
151
7. Cellular and Molecular Approaches to the

Investigation of Piscine Osmoregulation:
Current and Future Perspectives
Chris N. Glover
8. Osmoregulation and Fish Transportation
Paulo Csar Falanghe Carneiro,
Elisabeth Criscuolo Urbinati and Fabiano Bendhack
177
235
viii
Contents
9. Special Challenges to Teleost Fish Osmoregulation

in Environmentally Extreme or Unstable Habitats
Carolina A. Freire and Viviane Prodocimo
10. Energy Metabolism and Osmotic Acclimation in
Teleost Fish
Jos L. Soengas, Susana Sangiao-Alvarellos,
Ral Laiz-Carrin and Juan M. Mancera
11. The Renal Contribution to Salt and Water Balance
M. Danielle McDonald
249
277
309
12. Intestinal Transport Processes in Marine

Fish Osmoregulation
Martin Grosell
333
13. The Use of Immunochemistry in the Study of

Branchial Ion Transport Mechanisms
Jonathan Mark Wilson
359
14. Rapid Regulation of Ion Transport in

Mitochondrion-rich Cells
William S. Marshall
395
15. Control of Calcium Balance in Fish

Pedro M. Guerreiro and Juan Fuentes
427
16. Role of Prolactin, Growth Hormone, Insulin-like

Growth Factor I and Cortisol in Teleost Osmoregulation 497
Juan Miguel Mancera and Stephen D. McCormick
Index
517
Baldisserotto Bernardo
Departamento de Fisiologia e Farmacologia, Universidade Federal de
Santa Maria, 97105.900 Santa Maria, RS, Brazil.
E-mail: bernardo@smail.ufsm.br
Bendhack Fabiano
Pontifcia Universidade Catlica do Paran. Curitiba, Paran, Brazil.
E-mail: f.bendhack@pucpr.br
Brown J. Anne
School of Biosciences, University of Exeter, Exeter EX4 4PS, UK.
E-mail: J.A.Brown@exeter.ac.uk
Carneiro Paulo Csar Falanghe
Embrapa Tabuleiros Costeiros. Aracaju, Sergipe, Brazil.
E-mail: paulo@cpatc.embrapa.br
Cuesta Alberto
Fish Innate Immune System Group, Department of Cell Biology,
Faculty of Biology, University of Murcia, 30100 Murcia, Spain.
E-mail: alcuesta@um.es
Esteban M. ngeles
E-mail: aesteban@um.es
Ferreira Francesca W.
Departamento de Biologia e Qumica, Universidade Regional do
Noroeste do Rio Grande do Sul, 98700.000 Iju, RS, Brazil.
E-mail: piscis@unijui.tche.br
x List of Contributors
Flik Gert
Department of Organismal Animal Physiology, Faculty of Science,
Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen,
The Netherlands.
E-mail: g.flik@science.ru.nl
Freire Carolina A.
Departamento de Fisiologia, Setor de Cincias Biolgicas,
Universidade Federal do Paran (UFPR), Centro Politcnico, Bairro
Jardim das Amricas, Curitiba, PR, CEP 81531-990, Brazil.
E-mail: cafreire@ufpr.br
Fuentes Juan
Molecular and Comparative Endocrinology, Centre of Marine
Sciences, CCMAR, CIMAR Laboratrio Associado, University of
Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
E-mail: jfuentes@ualg.pt
Geven Edwin J.W.
The Netherlands.
E-mail: e.geven@science.ru.nl
Glover Chris N.
SCION, Te Papa Tipu Innovation Park, 49 Sala Street, Private Bag
3020, Rotorua, New Zealand.
E-mail: Chris.Glover@scionresearch.com
Grosell Martin
Rosenstiel School of Marine and Atmospheric Sciences, Division of
Marine Biology and Fisheries, University of Miami, 4600
Rickenbacker Causeway, 33145 Miami, Florida, USA.
E-mail: mgrosell@rsmas.miami.edu
Guerreiro Pedro M.
Molecular and Comparative Endocrinology, Centre of Marine
Sciences, CCMAR, CIMAR Laboratrio Associado, University of
Algarve, Campus de Gambelas, 8005-139 Faro, Portugal.
E-mail: pmgg@ualg.pt
xi
Hazon Neil
School of Biology, University of St Andrews, St Andrews KY16 8LB,
UK.
E-mail: nhl@st-andrews.ac.uk
Klaren Peter H.M.
The Netherlands.
E-mail: p.klaren@science.ru.nl
Kulczykowska Ewa
Department of Genetics and Marine Biotechnology, Institute of
Oceanology of Polish Academy of Sciences, Sopot, Poland.
E-mail: ekulczykowska@iopan.gda.pl
Laiz-Carrin Ral
Departamento de Biologa, Facultad de Ciencias del Mar y
Ambientales, Universidad de Cdiz, 11510 Puerto Real, Cdiz, Spain.
E-mail: raul.laiz@ca.ieo.es
Mancera Juan Miguel
Departamento de Biologa, Facultad de Ciencias del Mar y
Ambientales, Universidad de Cdiz, 11510 Puerto Real, Cdiz, Spain.
E-mail: juanmiguel.mancera@uca.es
Marshall William S.
Department of Biology, St. Francis Xavier University, P.O. Box 5000,
Antigonish, Nova Scotia, Canada B2G 2W5.
E-mail: bmarshal@stfx.ca
McCormick Stephen D.
USGS, Conte Anadromous Fish Research Center, Turners Falls, MA,
USA.
E-mail: steve_mccormick@usgs.gov
McDonald M. Danielle
Rosenstiel School of Marine and Atmospheric Science, University of
Miami, Miami, Florida, 33149-1098, USA.
E-mail: mcdonald@rsmas.miami.edu
xii
Meseguer Jos
E-mail: meseguer@um.es
Parra Jorge Erick Garcia
Departamento de Cincias Agrrias, Universidade Regional Integrada
do Alto Uruguai e das Misses Campus Santiago, 97700.000
Santiago, RS, Brazil.
E-mail: erickgarparr@yahoo.com.br
Prodocimo Viviane
Departamento de Fisiologia, Setor de Cincias Biolgicas,
Universidade Federal do Paran (UFPR), Centro Politcnico, Bairro
Jardim das Amricas, Curitiba, PR, CEP 81531-990, Brazil.
E-mail: vprodocimo@yahoo.com.br
Sangiao-Alvarellos Susana
Dr. Jos L. Soengas, Laboratorio de Fisioloxa Animal, Facultade de
Ciencias do Mar, Edificio de Ciencias Experimentais, Universidade de
Vigo, E-36310, Vigo, Spain.
E-mail: sangiao@uvigo.es
Soengas Jos L.
Laboratorio de Fisioloxa Animal, Facultade de Ciencias do Mar,
Edificio de Ciencias Experimentais, Universidade de Vigo, E-36310,
Vigo, Spain.
E-mail: jsoengas@uvigo.es
Urbinati Elisabeth Criscuolo
Universidade Estadual Paulista. Jaboticabal, So Paulo, Brazil.
E-mail: bethurb@caunesp.unesp.br
Wilson Jonathan Mark
Laboratrio de Ecofisiologia, Centro Interdisciplinar de Investigao
Marinha e Ambiental, Rua dos Bragas 289, 4050-123, Porto, Portugal.
E-mail: wilson_jm@cimar.org
+0)26-4

Immune and Osmoregulatory
System Interaction
Alberto Cuesta, Jos Meseguer and M. ngeles Esteban*
INTRODUCTION
Fish, a very diverse group, were the first vertebrates to present a complete
immune system about 450-500 million years ago. The innate and adaptive
immune responses that they display share many similarities with the
mammalian immune system. The fact that fish are poikilotherms and,
therefore, subjected to environmental temperature changes, makes their
adaptive responses very low and slow, which means that fish immunity is
highly dependent on the innate or non-specific immune response.
Therefore, study of the fish immune system is of great interest from the
phylogenetical viewpoint and it is in fish that the adaptive responses first
appeared. Moreover, the growth of aquaculture to provide food for the
human diet has prompted researchers to investigate immunological
techniques for the diagnosis and control of fish diseases, the development
of vaccines being the final goal (Ellis, 1988).
Authors address: Fish Innate Immune System Group, Department of Cell Biology, Faculty of
Biology, University of Murcia, 30100 Murcia, Spain.
*Corresponding author: E-mail: aesteban@um.es
2 Fish Osmoregulation
Fish live in a changeable environment and they must adapt to these
changes. As regards water salinity changes, fish are able to adapt to the
environmental salinity by the mechanism known as osmoregulation. In
general, fresh and marine water-living fish tend to maintain a net water
influx or efflux in order to keep the plasma osmolarity constant. The
organs involved in osmoregulation are the kidney, gills and intestine,
which have been morpho-functionally characterized in many fish species
(Meseguer et al., 1981; Lpez-Morales et al., 1990; Sakamoto et al., 2001;
Greenwell et al., 2003) and will be described in another chapter. Moreover,
when the organs are engaged in osmoregulation, other functions may be
affected. This happens, for example, in the case of immune functions.
The fish immune response is intended to eradicate an invading agent,
the antigen. It starts with the humoral and cellular components of the
innate immune system after coming into contact with structures of the
pathogen known as pathogen-associated molecular patterns (PAMPs),
which are common molecules not usually found in eucaryotic cells, such
as viral double stranded RNA, bacterial lipopolysaccharide (LPS) and
certain sugars. This response usually starts immediately and lasts several
hours. The antigen is then processed and presented to the adaptive
immune system components (B and T lymphocytes), which elaborate the
adaptive or specific response. This entire process takes several days but,
due to the lack of thermoregulation, the response achieved is never
comparable in terms of effectiveness with the mammalian response. The
control and integration of this immune response is carried out by
cytokines, which are mainly produced by lymphocytes and monocyte/
macrophages after stimulation. However, the immune response is also
modulated by many other intrinsic and extrinsic factors, including
environmental factors (temperature, salinity, photoperiod, etc.) and
physiological status (nutrition, age, reproductive cycle, hormonal balance,
stress, etc.).
Apart from the morphological features of the organs involved in
osmoregulation (Meseguer et al., 1981; Lpez-Morales et al., 1990), the
morpho-functional properties of the teleost immune system have been
characterized in our group (Esteban et al., 1989, 1998, 2001; Meseguer
et al., 1991, 1994, 1996; Mulero et al., 1994; Cuesta et al., 1999, 2002,
2003, 2004; Ortuo et al., 2000, 2002; Sepulcre et al., 2002; Chaves-Pozo
et al., 2003; Rodrguez et al., 2003; Salinas et al., 2005;). In this chapter,
we shall review the effect that salinity (as an environmental factor) may
Alberto Cuesta et al.
have on the fish immune responses, following by the importance and

magnitude of the osmoregulatory hormones (as an intrinsic factor) to
finally deal with the endocrine-osmoregulation-immunity interactions in
fish whose osmotic balance has been altered.
FISH IMMUNE SYSTEM ORGANIZATION
The fish immune system is as organized and complex as it is in mammals
(for reviews see Meseguer et al., 1995, 1996, 2002; Zapata et al., 1996;
Manning, 1998; Dixon and Stet, 2001; Evans et al., 2001; Magor and
Magor, 2001; Secombes et al., 2001). Due to variations in animal anatomy
and evolutionary position of fish, morpho-functional differences exist in
immune tissues and cells between fish and mammals.
Structure and Organization
The fish immune systemlike that of other vertebratesconsists of
physical barriers and immune organs. The first and principal barrier is the
skin, which together with the gills and gut, contains large amounts of
mucus. This mucus serves as an antimicrobial and antiparasitic barrier
because it contains highly active immune soluble factors such as lysozyme,
complement, C-reactive protein, lectins and immunoglobulins. Thus,
injuries in the barriers or the lack of mucus facilitate the entry of
pathogens into fish, where humoral and cellular immune effectors then
begin to play their part. The most characteristic difference from mammals
is the lack of bones, and therefore bone marrow, while the kidney is
divided into two functional parts: the pronephros (also called anterior or
head-kidney), which is the main haematopoietic organ in fish, and the
opisthonephros (called posterior or trunk kidney), which is mainly
dedicated to the excretory function. However, the immune functions are
conserved along the entire kidney. Apart from these, there are also small
batches of scattered immune cells in the gills and gut although, in general,
fish leucocyte types are quite similar to their mammalian counterparts,
except for granulocytes, while platelets are replaced by thrombocytes.
Innate Immune Response
Once the pathogen (bacteria, virus or parasite) has entered the fish, the
host elicits an inflammatory response involving humoral (complement,
lysozyme, C-reactive protein, lectins, etc.) and cellular (monocyte/
macrophages, granulocytes and lymphocytes) components of the innate
immune response. Complement and lysozyme are able to kill the
pathogens by puncturing their membranes. Among the cellular
mechanisms, phagocytosis and cytotoxicity are the main mechanisms
involved. Phagocytes (monocyte/macrophages and granulocytes) engulf
the pathogen and exert their lytic function through lysosomal enzymes
(peroxidases, etc.) and the production of reactive oxygen/nitrogen species
(O 2, H2O2 or NO). The nonspecific cytotoxic cells (NCC) are a
heterogeneous leucocyte population, functionally equivalent to the
mammalian natural killer (NK) cells, which mediate the cytotoxic activity
against tumor cells, virus-infected cells and parasitic protozoa. Apart from
complement and lysozyme, the humoral factors include C-reactive
protein, lectins, transferrin, anti-proteases, interferons and eicosanoids,
which form part of the innate response and combat the pathogen by means
of different mechanisms.
Adaptive Immune Response
The first functional studies carried out pointed to the presence of B and
T lymphocytes in fish because of the immune responses observed,
including specific cytotoxicity, antigen-specific antibody generation,
delayed hypersensitivity and graft rejection. The appearance of specific
antibodies directed against B or T cells and the development and
application of molecular biology tools have increased our understanding of
the adaptive immune responses in fish, while new findings in this area
tend to confirm the similarities with the mammalian adaptive immune
response, with a few exceptions. For example, the existence of rearranging
genes for immunoglobulin M (IgM), T-cell receptor (TCR) and major
histocompatibility (MHC) has been confirmed as has been the existence
of coreceptor molecules (CD3, CD4 and CD8). Further functional studies
will presumably demonstrate the great similarities existing between the
mammalian and fish adaptive immune systems from a molecular and
functional viewpoint.
Cytokines
Cytokines are immune system hormones. They are small polypeptides or
glycoproteins synthesized after leucocyte stimulation and even show
pleiotropic effects. Interleukin (IL)-1, IL-2, IL-3, IL-6, interferon (IFN),
tumor necrosis factor (TNF), transforming growth factor b1 (TGB-b1) and
chemokines are the main cytokines found in fish till date. The recent
availability of the cytokine gene sequences and ongoing production of
recombinant cytokines will throw light on their specific functions within
and outside the immune system.
Major Histocompatibility Complex (MHC)
MHCs are highly polymorphic cell surface proteins consisting of MCH
class I and class II glycoproteins. They belong to the immunoglobulin
superfamily of proteins and interact with the T-cell subsets through a
specific TCR, initiating the adaptive immune response. They are
responsible for presenting the antigen to the T lymphocytes and are
considered to be the link between the innate and adaptive immune
responses. Since they were first discovered by PCR techniques, the MHC
from several fish species have been cloned and studied from a genetic
point of view. They appear clustered in all vertebrates except for teleost
fish, where they are in different chromosomes and called MH receptors.
However, deeper knowledge of the involvement and functioning of the
MHC in the immune response is just emerging with the use of
recombinant MHC proteins and anti-MHC antibodies.
INFLUENCE OF ENVIRONMENTAL SALINITY ON FISH
IMMUNE RESPONSE
Salinity is one of the most important environmental factors for aquatic
organisms. In teleost fish, environmental salinity fluctuations trigger the
osmoregulatory response to compensate for such changes. However, other
physiological processes are also affected. For example, the immune
response and fish disease resistance is modulated by salinity, as has been
shown in several studies. Few experiments have examined the
immunological responses after salinity disturbances in fish, the innate
responses being the most analyzed thus far. The total circulating IgM
levels, which reflect the immune system status without exposing the fish
to a specific antigen (Yada et al., 1999), has been the most examined
immune parameter. On the other hand, cellular activities such as
phagocytosis, respiratory burst and cytotoxicity have hardly been
determined in the few investigations carried out. Future studies are
needed to establish the impact of salinity on the general immunological
status rather than the effect on an individual immune response.
Hyperosmotic adaptation has been mainly studied in salmonids
(Table 1.1). The first studies dealt primary immune responses in coho
salmon (Oncorhynchus kisutch), which were seen to decrease when the fish
entered seawater during smoltification (Maule and Schreck, 1987).
Brown trout (Salmo trutta) specimens transferred to seawater, on the other
hand, showed increased plasmatic lysozyme activity while the phagocytic
or natural cytotoxic activities of pronephric leucocytes increased or
remained unchanged, respectively (Marc et al., 1995). Specific antibody
titres to Yersinia ruckeri decreased in rainbow trout (Oncorhynchus mykiss)
7 days after transfer to 22 ppt salinity (Betoulle et al., 1995). On the other
hand, the circulating IgM level of trouts was unaffected 3 days after
transfer from freshwater (FW) to 12 ppt water, while the lysozyme activity
was 3.5-fold increased (Yada et al., 2001). The same fish were then
transferred from 12 ppt to 29 ppt salinity water and 24 h later they showed
the same level of IgM, while the lysozyme activity had further increased.
Peripheral blood leucocyte (PBL) production of superoxide (O 2),
measured by nitroblue tetrazolium (NBT) reduction, was greatly
increased. However, the same group did not detect any change in
plasmatic IgM, lysozyme activity or O2 production by PBLs in
Mozambique tilapia (Oreochromis mossambicus) transferred from FW to
35 ppt salinity water for more than 1 month, although head-kidney
leucocyte (HKL) production of O2 was increased (Yada et al., 2002).
Moreover, the authors conducted further research and described, for the
first time, the increase of PRL-R (prolactin receptor) mRNA expression in
leucocytes due to hypersaline adaptation. This PRL-R triggers a cascade
into the cell, leading to the cell responses, where activation of the immune
function is also produced. A recent study in Nile tilapia (Oreochromis
niloticus) has described the lethal effect of 35 ppt environments but
increased plasmatic IgM levels in specimens after 2 or 4 weeks of
adaptation to 12 or 24 ppt salinity (Dominguez et al., 2004). Although
both tilapia species, O. mossambicus and O. niloticus, have similar life
requirements, the differences observed could be due to several reasons.
Apart from the different salinity conditions (time and salinity stringency),
body size (50-100 or 18.2-21.7 g bw, respectively), diet ration or
temperature (24 and 28C, respectively) were also different. All these
parameters influence the osmoregulatory response and also the immune
response, as indicated above.
Few studies have evaluated the effects of environmental salinity
changes in marine fish species (Table 1.1). In winter flounder (Pleuronectes
14 or 28 ppt
33 ppt to 6 or 21ppt
FW to 12 or 29 ppt
FW to 22 ppt
FW to 35 ppt
Pleuronectes americanus
Mylio macrocephalus
Oncorhynchus mykiss
Maule and Schreck (1987)

Marc et al. (1995)
immune responses
lysozyme and phagocytosis
= cytotoxicity
blood thrombocytes in SW
phagocytosis
= IgM, lysozyme, O 2 in PBLs
anti-Yersinia ruckeri specific IgM
= IgM and lysozyme, - O 2 and PRL-R
expression in HKLs
IgM
peroxidases and ACH, = IgM
peroxidases, ACH, = IgM
IgM, = peroxidases and ACH
susceptibility to IPNV
resistance to Flavobacterium columnare
with the salinity increase
Chou et al. (1999)

Altinok and Grizzle (2001)
Domnguez et al. (2004)

Cuesta et al. (2005a)
Plante et al. (2002)

Narnaware et al. (2000)
Yada et al. (2001)
Betoulle et al. (1995)
Yada et al. (2002)
Reference
Immune parameter
FW, freshwater; SW, seawater; ppt, parts per thousand; PBLs, peripheral blood leucocytes; HKLs, head-kidney leucocytes; IPNV, Infectious pancreatic necrosis virus; PRL-R, PRL
receptor; ACH, alternative complement activity; , increase; , decrease; =, no effect.
Epinephelus sp.
Ictalurus punctatus
Acipenser oxyrinchus desotoi
Morone saxatilis
Carassius auratus
Oreochromis niloticus
Sparus aurata
FW to 12 or 24 ppt
40 to 6 ppt
40 to 12 ppt
40 to 55 ppt
33 ppt to 20 or 40 ppt
0, 1, 3 or 9 ppt
FW to SW
FW to SW
Oncorhynchus kisutch
Salmo trutta
Oreochromis mossambicus
Salinity acclimation
Species
Table 1.1 Effect of salinity disturbances on fish immune responses.
americanus), adaptation for 2 months to seawater (SW; 28.7 ppt) or
brackish water (BW; 14.7 ppt) completely abrogated the circulating
thrombocytes seen in SW and increased all the stress indicators (Plante
et al., 2002). Two studies have also been carried out in sparids. In gilthead
seabream (Sparus aurata), transfer from 40 ppt salinity to 55 ppt for
14 days increased the plasmatic IgM levels but did not affect the
alternative complement activity or the plasmatic peroxidases content
(Cuesta et al., 2005a). This finding agrees with the increased IgM levels
found in Nile tilapia (Dominguez et al., 2004) but contrasts with those
found in Mozambique tilapia and rainbow trout (Yada et al., 2001, 2002).
On the other hand, transfer from 40 ppt to 12 or 6 ppt salinity for 14 or
100 days decreased the peroxidase content and/or complement activity
but did not influence the circulating IgM levels. In the other study,
2-5 g bw black seabream (Mylio macrocephalus) specimens were kept at 33,
21 or 6 ppt salinity water for 72 days (Narnaware et al., 2000) and, while
the phagocytic activity of pronephric leucocytes increased in those fish
adapted to 6 or 21 ppt salinities compared to the fish maintained in fullseawater (33 ppt), the activity of spleenic leucocytes decreased. Moreover,
the authors demonstrated that the diet ration interacted with salinity in
the effect observed on the immune responses.
Many studies have demonstrated that the best culture conditions for
fish, both in aquaria and fish farms, are those in which the fish species are
in isoosmotic water. These conditions mean that the fish uses less energy
in osmoregulation and can redirect this energy towards other physiological
processes, such as growth or immune responses. In this way, the limited
data related with the defence mechanisms are presented. Mortalities of
1 g bw grouper fry (Epinephelus sp.) specimens transferred from 33 ppt
water to 20 or 40 ppt salinity water for 48 h increased (Chou et al., 1999).
Moreover, when they were exposed to IPNV either before or after the
salinity transfer, the mortality significantly increased, reaching 100% in
some cases. In another experiment, channel catfish (Ictalurus punctatus),
goldfish (Carassius auratus), striped bass (Morone saxatilis) and gulf
sturgeon (Acipenser oxyrinchus desotoi) were maintained in freshwater
(0 ppt), 1, 3 or 9 ppt salinity (Altinok and Grizzle, 2001). After
acclimation, they were exposed to an experimental infection with the
bacteria Flavobacterium columnare. None of the gulf sturgeons died, while
the mortality of the other fish species decreased with increased salinity,
with no mortality observed in the fish adapted to 3 or 9 ppt salinities.
However, most studies have analyzed or related salinity changes with the
pathogenic potential or survival of pathogens and not with the fish

defence. For example, Ichthyophthirius multifiliis strains isolated from
rainbow trout were susceptible to more than 5 ppt salinity (Aihua and
Buchmann, 2001), while the survival of the copepod Lerneaocera
branchialis, a parasite of the aquarium cod, is salinity restricted below 1620 ppt salinity (Knudsen and Sundnes, 1998). Apart from the direct effect
of salinity on the viability of pathogens, salinity seems to affect the PAMPs
because parasites incubated at different salinities change their virulence,
pathogenicity and even their adherence to the fish immune system
effectors (Bordas et al., 1996; Altinok and Grizzle, 2001; Nitzan et al.,
2004; Zheng et al., 2004). Results have demonstrated that salinity directly
affects the pathogenicity of virus, bacteria and parasites affecting the
subsequent clearance by the fish immune system.
Explanations of how the changes in osmotic pressure alter the
immune function of leucocytes are not consistent. The data suggest that
leucocytes, like the rest of the body cells, are affected by the osmotic
pressure. However, how and why they are shifted to inhibition or
activation after osmotic balance disruption remain unanswered. Although
the effect of osmoregulatory hormones on these cells (see below) is
supposed to be the key, some direct role must be operating in leucocyte
functioning. Perhaps, alterations in the water and ionic balance are
sufficient strong signals to change the immune response by themselves.
Furthermore, variations in plasmatic/seric levels might be attributed to the
increase/decrease of blood volume with the consequent dilution/
concentration, respectively, of humoral immune mediators. However, this
hypothesis cannot be supported in light of the ensuing results. These data
confirm the need for more in-depth studies into the role of salinity in the
immune system and disease resistance, and into the mechanisms involved.
OSMOREGULATORY HORMONESDO THEY CONTROL
THE IMMUNE SYSTEM?
It is well-known and assumed that fish present complex and bi-directional
endocrine-immune interactions (Weyts et al., 1999; Engelsma et al.,
2002). However, the mechanisms mediating such interactions are not well
studied, although they are supposed to be similar to those in mammals. We
shall now analyze endocrine-immune interactions, focusing on the
immunomodulatory potential of those hormones that play some osmoregulatory role. The major hormones involved in fish osmoregulation,
10
Fish Osmoregulation
namely PRL, GH and cortisol, have been shown to act as fish

immunomodulators. While PRL and GH have been found to increase
immune responses, cortisol is considered a stress hormone and plays an
antagonistic role. The effect of such hormones on the immune system was
first defined by studies involving fish transfer to hypo- or hyperosmotic
media, stressful situations and hypophysectomy and, lately confirmed by
in vitro and in vivo assays conducted with purified hormones. However,
more studies are needed to complete the information, regardless of their
exact effect on the immune response and disease resistance. Later
investigations tried to establish the precise osmoregulatory actions of
several other hormones, such as corticotropin, arginine, vasotocin,
epinephrine, norepinephrine, thyroid hormones (T3 and T4), estradiol,
aldosterone and natriuretic peptides (see Bentley, 1998). Future research
will tend to elucidate the role of the osmoregulatory hormones in the
immune system and will hopefully increase our knowledge concerning the
complex interactions between fish osmoregulation and immunity.
PRL and GH
These two pituitary hormones have a demonstrated immunostimulatory
role in fish. First evidence pointed in this direction after the effects on the
immune system in hypophysectomized fish were studied. In this sense,
killifish (Fundulus heteroclitus) showed an important reduction in the
number of circulating leucocytes (Pickford et al., 1971). Removal of the
pituitary in rainbow trout decreased the levels of plasmatic IgM, Igsecreting leucocytes in head-kidney and blood, as well as O2 production
by HKLs (Yada et al., 1999; Yada and Azuma, 2002). On the other hand,
lysozyme activity, the total number of leucocytes, O2 production by PBLs
and Ig-secreting cells in thymus and spleen were unaffected. In
hypophysectomized O. mossambicus, however, neither plasmatic IgM level
nor the lysozyme activity was modified, while O 2 production by HKLs was
depressed (Yada et al., 2002). These same experiments also demonstrate
the reversion of the immune response caused by hypophysectomy after
exogenous PRL or GH administration.
In vitro or in vivo treatment of fish with PRL or GH (either from fish,
mammalian or recombinant source) enhances the humoral (IgM level as
well as complement and lysozyme activities) and cellular (mitogenesis,
phagocytosis, cytotoxicity and respiratory burst) responses of the fish
immune system, as well as disease resistance (Table 1.2). They exert their
11
Table 1.2 Effects of principal osmoregulatory hormones (PRL, GH and cortisol) on fish
immune responses.
Hormone Effect
PRL
GH
Cortisol
Species
Reference
mitogenesis
Oncorhynchus keta
O. mykiss
Sakai et al. (1996b)

Yada et al. (2004a)
phagocytosis
Sparus sarba
respiratory burst
O. mykiss
Sakai et al. (1996c)

Yada et al. (2002)
lysozyme activity
O. mykiss
Yada et al. (2001, 2004b)
allograft rejection
Fundulus grandis
Nevid and Meier (1995)
IgM levels
O. mykiss
Yada et al. (1999)
IgM levels
Sparus aurata
Cuesta et al. (2005b)
lymphopoiesis
S. aurata
S. sarba
Calduch-Giner et al. (1995)

phagocytosis
O. mykiss
Oncorhynchus keta
S. aurata
Sakai et al. (1995, 1996c, 1997)

Calduch-Giner et al. (1997)
mitogenesis
O. keta
O. mykiss

Yada et al. (2004a)
cytotoxic activity
O. mykiss
Kajita et al. (1992)
IgM levels
O. mykiss
Yada et al. (1999)
lysozyme activity
O. mykiss
Yada et al. (2004b)
haemolytic activity
O. mykiss
Sakai et al. (1996a)
disease resistance
O. keta
Sakai et al. (1997)
respiratory burst
O. mykiss
O. keta
Dicentrarchus labrax
Sakai et al. (1995, 1996c)

Kitlen et al. (1997)
Yada et al. (2001)
Sakai et al. (1996b, 1997)
Muoz et al. (1998)
Yada et al. (2002)
IgM levels
Sparus aurata
circulating
lymphocytes
O. kisutch
McLeay (1973)
Salmo trutta
Ictalurus punctatus
S. salar
Cyprinus carpio
Pickering (1984)
Ellsaesser and Clem (1987)
Espelid et al. (1996)
Wojtaszek et al. (2002)
Pleuronectes platessa
Grimm (1985)
leucocyte
mitogenesis
(Table 1.2 contd.)
12
Fish Osmoregulation
(Table 1.2 contd.)
Oncorhynchus kisutch
Ictalurus punctatus
Salmo salar
Cyprinus carpio
O. mykiss
O. mykiss cell line RTS11
circulating/production O. mykiss
IgM
C. carpio
O. kisutch
Pleuronectes americanus
O. tshawytscha
O. masou
Tripp et al. (1987)

Ellsaesser and Clem (1987)
Espelid et al. (1996)
Weyts et al. (1997)
Yada et al. (2004)
Pagniello et al. (2002)
Anderson et al. (1982)
Hou et al. (1999)
Ruglys, (1985)
Saha et al. (2004)
Maule et al. (1987)
Tripp et al. (1987)
Carlson et al. (1993)
Milston et al. (2003)
Nagae et al. (1994)
phagocytosis
C. carpio
Oreochromis niloticus
Sparus aurata
C. carpio
C. auratus macrophage
cell line
Law et al. (2001)

Law et al. (2001)
Esteban et al. (2004)
Watanuki et al. (2002)
Wang and Belosevic (1995)
chemotaxis
cell line
respiratory burst
S. aurata
C. carpio

Kawano et al. (2003)
Stave and Roberson (1985)
Morone saxatilis
NO production
C. carpio
cell line

immune genes
expression
O. mykiss
Zou et al. (2000)
C. carpio
Saeij et al. (2003)
circulating IgM
S. aurata
apoptosis
C. carpio
O. mykiss
O. mossambicus
Weyts et al. (1997, 1998a)

Saha et al. (2003)
Yada et al. (2004)
Bury et al. (1998)
C-reactive protein
P. platessa
White and Fletcher (1985)
allograft rejection
Fundulus grandis
Nevid and Meier (1995)

(Table 1.2 contd.)
13
(Table 1.2 contd.)
pathogen
susceptibility
Prevents apoptosis in
neutrophils
Prevents stress
immunodepression
= cytotoxicity
O. mykiss
Kent and Hedrick (1987)
S. salar
S. trutta
Salvelinus alpinus
Wiik et al. (1989)

Harris et al. (2000)
C. carpio
Weyts et al. (1998b)
O. mykiss
Narnaware and Baker (1996)
S. aurata
, increase; , decrease; =, no effect; NO, nitric oxide.
actions after engaging their specific receptors in the cells. Both hormones
belong to the cytokine/haematopoietin family, while their receptors belong
to the class I superfamily of cytokine receptors (see Clevenger et al., 1998;
Moutoussamy et al., 1998; Power, 2005). Evidence of the mRNA
expression of PRL and GH, as well as their respective receptors, have been
documented in lymphoid organs and isolated leucocytes in several
teleosts, including tilapia, rainbow trout, gilthead seabream, orangespotted grouper (Epinephelus coioides), coho salmon, goldfish, masou
salmon (Oncorhynchus masou), japanese flounder (Paralichtys olivaceus)
and black seabream (Acanthopagrus schlegeli) (Weigent et al., 1988; Sandra
et al., 1995; Mori and Devlin, 1999; Santos et al., 1999, 2001; Yang et al.,
1999; Prunet et al., 2000; Tse et al., 2000, 2003; Higashimoto et al., 2001;
Lee et al., 2001; Yada and Azuma, 2002; Yada et al., 2002; Fukada et al.,
2004; Zhang et al., 2004; Power, 2005). In mammals, lymphocytes and
macrophages are the leucocyte-types that express both hormones and
hormone-receptors, and the pattern might be the same in fish. Thus,
leucocyte activation may not only be due to pituitary-secreted PRL and
GH but may also be caused by the self-produced hormones. In this way,
both hormones could be considered as cytokines, as they are in mammals,
and autocrine and paracrine actions within the immune system are
actually under consideration.
Receptors for fish PRL and GH (PRL-R and GH-R respectively) show
the conserved motifs of the cytokine-receptor family. Thus, fish PRL-R is
only present in the long and intermediate forms with the conserved motif
WSXWS (Trp-Ser-Xaa-Trp-Ser) in the extracellular domain, while in the
GH-R the conserved motif found is Y/FGEFS (Tyr/Phe-Gly-Glu-Phe-Ser).
14
Fish Osmoregulation
In both receptors, the single transmembrane region is followed by a

cytoplasmic region containing conserved proline-rich motifs (box-1 and
box-2) and phosphorylable residues. Similarly to mammals, PRL-R and
GH-R binding to their respective hormones on fish leucocytes probably
involves the participation of the Jak/STAT activation pathway, although
there are, to date, no specific data to support this interaction in fish.
However, apart from the conservation of box-1 and box-2 motifs in the
receptors, the presence of the Jak/STAT pathway in fish leucocytes has
been confirmed (Jaso-Friedmann et al., 2001; Santos et al., 2001; Fukada
et al., 2004; Cuesta et al., 2005c). Another striking point is the crossinteractions between forms of PRL and GH-R (Auperin et al., 1995;
Sandra et al., 1995; Shepherd et al., 1997). In tilapia, the PRL177 is able
to bind the GH-R and, could lead to a stimulation of leucocytes mimicking
the effects due to either PRL or GH. These two findings are probably the
most valuable for deciding future directions that should be taken. It is
imperative to distinguish between the effects due to the PRL form or GH,
as well as to identify which hormone receptor is responsible for the
immunostimulation achieved. Molecular approaches can hopefully be
conducted in order to finally clarify the molecular interactions and
involvement of the Jak/STAT activation cascade in the modulation of the
immune system by these pituitary hormones.
Cortisol
The principal inter-renal gland-produced hormone, cortisol, is considered
the stress hormone but it is also involved in osmoregulation and the
immune function. Although the immunosuppressive effects observed after
stress are attributed to high levels of circulating cortisol (reviews of Balm,
1997; Wendelaar Bonga, 1997; Pickering, 1998; Harris and Bird, 2000a),
we will only focus on the investigations directed at evaluating the impact
of exogenous in vitro or in vivo administration of cortisol on the fish
immune system (Table 1.2). In this sense, most of the studies conducted
demonstrate that cortisol treatment by itself decreases the fish immune
functions, as does stress. However, differences in treatment (cortisol
concentration and time), fish species, leucocyte source and immune
parameter measured may affect the results observed. Thus, several papers
suggest that cortisol is not the mediator of the stress effects and point to
the need for more and deeper studies need to be done before any general
rule can be assumed. For example, Narnaware and Baker (1996)
15
demonstrated that trout injected with cortisol recovered from the

immunosuppressive effects after an acute stress. They found decreased
levels of circulating lymphocytes and phagocytic activity in stressed fish.
These immunological changes were abrogated and restored in those fish
injected with physiological concentrations of cortisol. As a hypothesis,
authors thought that cortisol might inhibit the release of catecholamines,
which would be directly responsible for the stress-response in some way.
Another explanation could be that cortisol mediates the expression of
adhesion molecules in leucocytes and therefore their trafficking. So, as in
mammals (Chung et al., 1986), cortisol administration may impair
lymphocyte recruitment in the lymphoid tissues, while circulating
granulocyte and/or macrophage numbers may be increased (Ellsaesser and
Clem, 1987; Narnaware and Baker, 1996; Ortuo et al., 2001; Wojtaszek
et al., 2002). If these circulating phagocytes are the active cells from the
spleen or pronephros, the phagocytic activity of the remaining phagocytes
must be inhibited, which would agree with most studies. Weyts et al.
(1998a,b) found more striking data. They demonstrated that cortisol did
not induce apoptosis in circulating T lymphocytes and thrombocytes but
did so in B lymphocytes. Moreover, circulating neutrophils treated with
high cortisol levels were protected from apoptosis, making these
leucocytes more able to attack the pathogens entering the body. Moreover,
cortisol did not inhibit their respiratory burst, which could be essential for
survival since they form part of the first line of defence. Esteban et al.
(2004) also investigated the cortisol effect on the gilthead seabream
immune response. In vitro, pharmacological dosages of cortisol decreased
the phagocytosis of head-kidney leucocytes but unaffected the respiratory
burst and cytotoxicity. On the other hand, in vivo administration of
cortisol (reaching plasmatic levels similar to those after acute stress)
increased the circulating IgM levels and left unaltered the complement
activity (Cuesta et al., 2005b). This variability in the data concerning the
immunosuppressive effects of cortisol, as well as contrary findings, should
stimulate researchers into conducting more investigations in this field to
ascertain how cortisol acts and how influences the fish immune system.
To date, cortisol synthesis has only been described in the interrenal
gland and not in the leucocytes. On the other hand, the expression of
glucocorticoid (GR) and mineralocorticoid (MR) receptors in lymphoid
organs has been mentioned in several fish species, including rainbow
trout, carp, coho salmon, tilapia and Astatotilapia burtoni (Maule and
16
Fish Osmoregulation
Schreck, 1990; Ducouret et al., 1995; Tagawa et al., 1997; Weyts et al.,
1998c; Colombe et al., 2000; Bury et al., 2003; Greenwood et al., 2003).
However, functional data support the notion that fish leucocytes contain
MR and GR, as do their mammalian counterparts, although the specific
cell-types expressing them are not known. Furthermore, the effects
described for cortisol on the immune response are mimicked by the agonist
dexamethasone and abrogated by the blocking agents cycloheximide or
RU486 (Weyts et al., 1998b; Law et al., 2001; Pagniello et al., 2002;
Esteban et al., 2004). Although there are evident analogies between fish
and mammals as regards the receptor activation cascade and effects upon
the immune related genes further studies are needed to clarify the effects
of cortisol on leucocytes at molecular level.
Other Hormones
Many other fish hormones play some osmoregulatory role either by direct
or indirect action. For example, they may affect the release of PRL, GH or
cortisol, and modify Na+-K+ ATPse activity, etc. (see Bentley, 1998).
However, the effects of these hormones on the fish immune system have
not been studied in any depth. Thus, melanocyte-stimulating hormone
(a-, b-, g- and d-MSH), b-endorphin (b-EP) or adrenocorticotropin
hormone (ACTH) are produced in fish leucocytes and are therefore
supposed to have autocrine and paracrine actions (Ottaviani et al., 1995;
Balm et al., 1997; Amemiya et al., 1999; Arnold and Rice, 2000). MSHs
and b-EP are able to stimulate leucocyte proliferation and phagocyte
functions, including phagocytosis, respiratory burst and the release of
macrophage-stimulating factor (Harris and Bird, 1997, 1999, 2000b;
Takahasi et al., 1999; Watanuki et al., 1999, 2000, 2003). ACTH, on the
other hand, inhibits circulating leucocyte numbers and lymphocyte
mitogenesis while activating phagocytosis and respiratory burst activity
(McLeay, 1973; Bayne and Levy, 1991; Weyts et al., 1999). Another
melanotropin, the melanin-concentrating hormone (MCH), has been
shown to affect fish immune responses in a similar way to the MSHs
(Harris and Bird, 1997, 1999, 2000b; Watanuki et al., 2003). Some sexual
hormones have been found to be involved in osmoregulation and also
affect the immune response. Estradiol, progesterone, testosterone or
11-ketotestosterone have been found to influence the immune response
negatively, while few assays describe immunoactivation (Harris and Bird,
2000a; Law et al., 2001; Watanuki et al., 2002; Chaves-Pozo et al., 2003;
17
Saha et al., 2004; Cuesta et al., in press). In the future, the specific role of
these hormones on osmoregulation and immunity should be assayed in
order to ascertain and clarify their pleiotropic functions in teleost fish and,
more specifically, in osmoregulation and immunity.
ROLE OF FISH CYTOKINES IN THE ENDOCRINE
SYSTEM
So far, there is no information about the effect of fish cytokines on
osmoregulation. However, in mammals, bi-directional cross talk between
endocrine and immune systems has been described. Mammalian pituitary
cells, for example, are known to produce several cytokines (IL-1, IL-6,
TNF and IFN) and respond to them by means of their specific receptors
(see Thurnbull and Rivier, 1999; Engelsma et al., 2002). Moreover, the
administration of TNF and IL-6, but especially IL-1, stimulates the HPAaxis to produce ACTH, CRH and GC during infection, inflammation and
stress in mammals. Taking into account these data and similarities
between the mammalian HPA-axis with its fish HPI-axis counterpart, bidirectionality could also be assumed in fish. Although in a first step, few
available data on fish confirm this parallelism and the recent availability
of cytokine sequences points to promising future findings.
IL-1b gene expression is found in brain and in the pituitary of teleost
fish (Engelsma et al., 2001; Pelegrin et al., 2001). First studies
demonstrated that cortisol inhibits IL-1b mRNA levels in trout (Zou et al.,
2000) and carp (Engelsma et al., 2001), perhaps because the hormone
inactivates NF-kB, leading to no cytokine synthesis as occurs in mammals
(McKay and Cidlowski, 1999). Moreover, recombinant fish IL-1b triggers
the liberation of a-MSH and b-endorphin from pituitary in carp (see
Engelsma et al., 2002). In trout, recombinant IL-1b injection increased
circulating levels of cortisol (Holland et al., 2002), also demonstrating that
the effect was mediated by interaction with the hypothalamus-pituitary
gland. It is known that dexamethasone blocks endogenous ACTH
liberation with subsequent inhibition of cortisol release. Trout treated
with IL-1b and dexamethasone together did not show increased cortisol
levels. These results are also in agreement with the finding of IL-1 receptor
expression in brain and pituitary cells (Holland et al., 2002). The scant
results are promising and future studies concerning endocrine-immune
system interactions, as well as with other systems, need to be conducted.
18
Fish Osmoregulation
INTERACTIONS BETWEEN OSMOREGULATORY AND

IMMUNE RESPONSES
Many studies are confined to describing individual effects of treatment on
a specific response. However, integrative analysis of what happens
throughout the animal physiology after a given treatment represents the
most valuable studies but at the same time, the most difficult to achieve.
Thus, information about growth, stress, metabolism, hormonal status,
osmoregulation or immunity after treatment or commonly occurring
situations in fish farming, such as salinity disturbance, will hopefully be of
help. All these isolated data are in the process of being collated and future
multidisciplinary studies will ascertain why and how they interact, as well
as the consequences to the animal in terms of growth, quality, disease
resistance and environmental impact.
Although effects are inter-specific, hypophysectomized fish show a
lack of osmoregulation and a decreased immune response. In particular,
hypophysectomized trouts and tilapia have shown reduced values of some
immunological parameters (Yada et al., 1999, 2002a,b) although both
osmoregulatory and immune functions were restored after administration
of exogenous PRL or GH, indicating their central role in both systems,
although more studies should be carried to identify other potential
mediators (Yada et al., 1999). Many fish, including salmonids, tilapia and
sparids, have shown increased pituitary expression of PRL mRNA
accompanied by higher circulating levels of PRL after transfer to
hypoosmotic waters (Yamauchi et al., 1991; Mancera et al., 1993a; Martin
et al., 1999; Laiz-Carrin et al., 2005). However, transfer of fish from SW
to lower salinity media decreased the phagocytic activity in black
seabream, while in gilthead seabream the peroxidases content decreased
and plasmatic IgM levels remained unaffected (Narnaware et al., 2000;
Cuesta et al., 2005a). The complement activity of gilthead seabream was,
on the other hand, differently affected and depended on the adaptation
period. However, gilthead seabream is the only described case in which the
increase of PRL, either by exogenous administration or as a result of
transfer to hypoosmotic media, produces similar effects, that is,
suppression of the immune system (Cuesta et al., 2005a,b). Following with
this idea, Yada et al. (2002) found that the hypoosmoregulatory and
immunostimulant actions of PRL are drastically opposed, suggesting that
the role of PRL in osmoregulation and immunity are independent.
Unfortunately, there is little information about the expression of the PRL
19
and PRL-R genes in lymphoid tissues and leucocytes and about whether
they are modulated or not by plasmatic PRL levels. Yada et al. (2002)
demonstrated that head-kidney leucocytes from tilapia increase PRL-R
mRNA expression after transfer from FW to SW. This finding correlates
well with the studies describing increased immune responses after
hyperosmotic adaptation (see Table 1.1) and could explain part of the
immunostimulation produced after hyperosmotic adaptation. Moreover,
although the transfer from FW to SW decreases PRL release, favouring
acclimation to saline conditions, the affinity and capacity of PRL-R is
rapidly increased and maintained for several weeks (Auperin et al., 1995;
Sandra et al., 2001). Furthermore, the expression of mRNA coding for the
PRL-R gene was unaffected in head-kidney leucocytes or in the gills of
hypophysectomized tilapia specimens (Auperin et al., 1995; Yada et al.,
2002). These observations indicate that factors other than the presence
and abundance of pituitary hormones might be controlling the expression
of PRL-R, especially in lymphoid tissues, and, by extension, the immune
function. Perhaps, paracrine actions of the leucocyte-produced PRL could
be the key and need to be investigated.
GH, on the other hand, is clearly involved in hyperosmotic adaptation
in salmonids but behaves differently, depending on the species and salinity
in non-salmonids (Mancera and McCormick, 1998). The correlation was
best observed in brown trout, which showed increased levels of plasmatic
GH after transfer from FW to SW, along with increased lysozyme activity
and phagocytosis (Marc et al., 1995). Increased GH levels, as a result of
hyperosmotic environment adaptation or exogenous administration, tend
to correlate well with increased immune responses (Tables 1.1 and 1.2).
However, trout exhibited lower specific antibody titres in SW than in FW
(Betoulle et al., 1995). The total IgM levels were unaffected or increased
in several fish species adapted to hyperosmotic environments (Yada et al.,
2001, 2002; Dominguez et al., 2004; Cuesta et al., 2005a). On the other
hand, seabream injected with GH showed lower values of this parameter
(Cuesta et al., 2005b). While the total pool of circulating IgM might be
augmented by increases in salinity, the production of specific IgM is
inhibited because one or more steps in the generation of specificity
(antigen uptake, processing and presentation, selection of a specific IgMproducing lymphocyte B or IgM production) may be affected. Superoxide
anion production was decreased in HKLs but not in PBLs after
hypophysectomy, indicating differences in hormonal control in the
20
Fish Osmoregulation
different leucocyte sources (Yada and Azuma, 2002; Yada et al., 2002).
Similarly, GH injection restored IgM production in hypophysectomized
trouts (Yada et al., 1999). The injection of GH, together with
hyperosmotic adaptation, failed to over-stimulate IgM production and
lysozyme activity compared with that observed in fish only adapted to
higher salinity, while superoxide production by PBLs increased (Yada et al.,
2001). Unfortunately, there are no studies concerning the role of osmotic
change in the expression of GH-R. More and deeper analyses need to be
carried out regarding GH-R expression in different physiological
situations, since GH-R has been shown to interact with PRL. One form
of the tilapia PRL (PRL177) is structurally similar to GH and is therefore
recognized by GH-R, while PRL-R does not bind GH (Auperin et al.,
1995; Sandra et al., 1995; Shepherd et al., 1997). Strikingly, this explains
the increased PRL-R in SW-adapted fish and the increased immune
response after GH administration or hyperosmotic adaptation. Future
investigations to identify the involvement of PRL/GH-R interactions in
FW or SW adaptation will be welcome.
Salinity disturbance could also be considered stressful for fish,
although some data such a claim difficult to establish. Cortisol plays an
important role in hyperosmotic adaptation though it can also promote
adaptation to hypoosmotic environments, depending on the fish species
(Mancera et al., 1993b, 2002; Morgan and Iwama, 1996; Eckert et al.,
2001; McCormick, 2001; Laiz-Carrin et al., 2003). The circulating
cortisol levels reached after fish received implants of exogenous cortisol
are similar to those found in fish adapted to hyperosmotic environments
(Morgan and Iwama, 1996). Apart from its role in osmoregulation, cortisol
is considered responsible for the inhibition of the immune system in stress
situations. However, multiple interactions between endocrine-immune
systems must be operating. Most of the studies based on the effect of
cortisol on the immune response describe its depressive role (Table 1.2)
while, experiments in which fish are adapted to hyperosmotic media and
are therefore supposed to have elevated cortisol levels, generally point to
activation of the immune responses (Table 1.1). Thus, there are enough
data, even in the same fish species, to contradict the inhibitory hypothesis.
Everything depends on the response measured and the tissue or cells used
for immunologic determinations. Transfer or adaptation to hypersaline
waters of coho salmon depressed the innate immune system (Maule and
Schreck, 1987) while in rainbow trout the production of specific
21
antibodies was decreased (Betoulle et al., 1995). In many other studies the
immune responses increased. As regards humoral factors, circulating total
IgM levels are not affected in SW-adapted salmonids, which could be due
to the decrease in circulating lymphocytes. However, in gilthead
seabream, the IgM levels were increased both in hypersaline-adapted and
cortisol-implanted specimens (Cuesta et al., 2005a,c). The activity of
lysozyme, which is produced and released by mature monocyte/
macrophages and granulocytes, is increased after hyperosmotic
adaptation. On the other hand, plasmatic cortisol impairs bloodcirculating lymphocytes and their functioning (mitogenesis and the
production of specific IgM) and, at the same time, they increase their
susceptibility to die by apoptosis. Moreover, cortisol increases leucocyte
trafficking and the number of phagocytic cells in the blood. The
consequences of this cell extravasation could be an increase in lysozyme
activity in the serum, the levels of free-oxygen radicals and allograft
rejection due to mobilization of active leucocytes (Marc et al., 1995; Nevid
and Meier, 1995; Ortuo et al., 2001; Yada et al., 2001). Moreover, some
of these data are supported by the finding that cortisol protects
neutrophils against apoptosis (Weyts et al., 1998b). Another consequence
is the clearance of phagocytic cells from the lymphoid organs such as headkidney and spleen. This result in myeloid precursors dividing and
differentiating faster and therefore the monocyte/macrophages and
granulocytes present will be more immature and, obviously, their immune
responses (phagocytosis, respiratory burst, etc.) will be negatively affected.
The intention behind this impairment of the defence mechanisms in
organs such as the head-kidney and increase in some of the blood
leucocytes is clear: the availability of active circulating phagocytes to
overcome a possible pathogen invasion in altered fish homeostasis
(salinity shock or other stressful situation). However, and unfortunately,
the animal may not be able to overcome the pathogen as demonstrated in
several studies (Kent and Hedrick, 1987; Wiik et al., 1989; Chou et al.,
1999; Harris et al., 2000). Furthermore, cortisol has been proposed as a
candidate for overcoming the stress situations. Thus, trouts injected with
cortisol were protected from immunosuppressive effects due to stress
(Narnaware and Baker, 1996). Cortisol injection also decreased the
expression of stress-related immune genes in the common carp (Kawano
et al., 2003). All these data suggest that cortisol plays a dual role in the
immune system, as it does in the osmoregulatory response, which depends
on the fish species studied and the particular parameter determined.
22
Fish Osmoregulation
As commented above, other hormones, among their pleiotropic

actions, may also be involved in osmoregulation and immunity. However,
their effect on fish osmoregulation or immunity is not clear and hopefully
will be the target of future research. The presence and expression of
hormones and their receptors in endocrine and immune relevant cells as
well as the mechanisms and possible interactions need to be clarified.
CONCLUDING REMARKS
Investigations demonstrate and confirm the cross-regulation and
interaction between osmoregulation and immunity in teleost fish.
However, the mechanisms by which salinity and osmoregulatory
hormones up- or down-regulate the immune responses are not
understood. The presence of hormone receptors in fish leucocytes seems
to be essential but there are no data confirming this hypothesis. In this
sense, experiments using receptor blockers together with osmotic shock or
hormonal treatment are needed. So far, variations in hormone receptor
affinity or number after hypo or hyperosmotic adaptation have been
scarcely reported (Sandra et al., 2001; Dean et al., 2003). Moreover, the
autocrine and paracrine actions of the hormones in the lymphoid tissues
need to be evaluated. However, it seems evident that many other factors
and interactions are also active.
Finally, the role of cytokines in osmoregulatory and endocrine organs
need to be understood before we can understand these interactions.
Again, the finding that pituitary cells are able to produce cytokine and
their receptors opens an interesting investigation line. Obviously, the
paracrine and autocrine control of the synthesis of hormones and
consequently in the hormonal control of the osmoregulatory process must
be determined.
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+0)26-4
The Involvement of the Thyroid

Gland in Teleost Osmoregulation
Peter H.M. Klaren*, Edwin J.W. Geven and Gert Flik#
INTRODUCTION
It is not our goalnor is it desirableto provide a review of fish thyroid
physiology here. Indeed, others have comprehensively and authoritatively
treated the physiology of the piscine thyroid gland and thyroid hormones
(Eales and Brown, 1993; Leatherland, 1994). We have chosen to describe
some thyroidological aspects concisely, aiming to identify less wellinvestigated areas of piscine thyroidology. Specifically, we wish to focus
briefly on the teleost hypothalamus-pituitary gland-thyroid axis, the
regulation of which allows bidirectional communication with the teleost
stress axis. We shall also discuss the presence of heterotopic thyroid
follicles in osmoregulatory organs. With this contribution, we wish to
Authors address: Department of Organismal Animal Physiology, Faculty of Science, Radboud

University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands.
Corresponding authors: E-mail: *p.klaren@science.ru.nl; #g.flik@science.ru.nl
36
Fish Osmoregulation
suggest the use of parameters other than thyroid gland morphology and
plasma thyroid hormone concentrations, and to further the investigation
of the involvement of thyroid hormones in osmoregulation and other
aspects of fish physiology.
THYROID HORMONE BIOSYNTHESIS AND PLASMA
TRANSPORT
Biosynthesis
Transcellular iodide transport by the thyrocyte is established by a
concerted action of basolaterally and apically located transporters. A
Na+/I symporter (NIS) (Dai et al., 1996) located in the basolateral
membrane allows the thyrocyte to load systemic iodide from the
circulation. NIS activity is inhibited directly by thiocyanate and
perchlorate (Van Sande et al., 2003), and indirectly by ouabain (Ajjan
et al., 1998) through the inhibition of Na+, K+-ATPase and the
subsequent collapse of the transmembrane Na+ gradient which drives
iodide transport. The novel Cl/anion exchanger pendrin (Scott et al.,
1999) is believed to constitute the apical iodide extrusion pathway (Bidart
et al., 2000; Royaux et al., 2000; Yoshida et al., 2002). Recently, a human,
perchlorate-sensitive, apical iodide transporter (hAIT), with high
homology to hNIS, has been proposed as an alternative transport
mechanism (Rodriguez et al., 2002) (reviews on thyroid gland iodine
metabolism: Spitzweg et al., 2000; Dunn and Dunn, 2001).
To date, no piscine homologues of the transporters involved in
thyrocyte transcellular iodide movement have been identified. Even so,
the presence of NIS in teleosts can be inferred from the reduced
accumulation of radioiodide by zebrafish (Brown, 1997) and Mozambique
tilapia (Oreochromis mossambicus) (our unpublished results) after
treatment with the goitrogen perchlorate, and, in agnathans, from the
drastically decreased thyroidal iodide uptake and plasma thyroid hormone
levels in larval lampreys treated with different goitrogens (Manzon et al.,
2001; Manzon and Youson, 2002).
Thyroglobulin is a large (ca. 660 kDa) homodimeric glycosylated
protein synthesized by the thyrocyte and secreted into the follicular lumen
where it comprises a major component of the colloid. Thyroglobulins were
identified in cyclostomes and elasmobranchs ( Suzuki et al., 1975; Monaco
Peter H.M. Klaren et al.
37
et al., 1976, 1978) and teleosts (Kim et al., 1984; Baumeister and Herzog,
1988). In the afollicular endostyle of larval cyclostomes, thyroglobulin was
found to be localized in the cytoplasm and associated with the apical
membrane of a subpopulation of cells (Wright et al., 1978a,b).
Thyroid peroxidase (TPO) is an integral protein of the thyrocyte
apical membrane. The enzymes catalytic site is located extracellularly and
faces the follicular colloid where it catalyzes the oxidation of iodide (I) to
iodonium (I+). TPO further catalyzes iodine organification, which
involves the substitution of hydrogen atoms at the 3- and 5-positions of
the phenolic ring of tyrosine residues in thyroglobulin with iodonium. This
results in the formation of mono- (MIT) and diiodotyrosines (DIT). TPO
also catalyzes the coupling of iodotyrosine residues to form the
iodothyronines thyroxine, T4 (3,5,35-tetraiodothyronine), by the
coupling of two DIT molecules, and some 3,5,3-triiodothyronine, T3
(MIT + DIT). Organification and iodothyronine formation are inhibited
by the TPO-inhibitors 6-n-propyl-2-thiouracil (PTU) and methimazole
(MMI), which are clinically used as thyrostatics to treat hyperthyroidism.
No piscine TPO homologues have been identified so far, but treatment of
fishes with PTU or MMI successfully induces hypothyroidism (De et al.,
1989; Van der Geyten et al., 2001; Varghese et al., 2001; Elsalini and Rohr,
2003) from which the presence of TPO can be inferred.
Thyroglobulin is stored in the follicular lumen where it forms the
major constituent of the colloid. Micropinocytosis and colloid resorption
produce endosomes that fuse with primary lysosomes to form fagosomes.
Endo- and exopeptidase activities hydrolytically digest thyroglobulin to
smaller dipeptide fragments with the concomitant release of
iodothyronines. The thyroid hormones are secreted across the basolateral
membrane of the thyrocyte through an as yet unknown mechanism, but
which would most likely include a membrane transport protein.
PLASMA TRANSPORT AND CELLULAR UPTAKE OF
THYROID HORMONES
Native iodothyronines are lipophilic, and binding proteins facilitate
convective plasma transport of thyroid hormones. In mammals, thyroid
hormones are bound to (in the order of decreasing T4-binding affinities):
thyroxine-binding globuline (TBG), transthyretin (TTR, previously
designated thyroxine-binding prealbumin or TBPA) and albumin
38
Fish Osmoregulation
(Schreiber and Richardson, 1997; Schussler, 2000). Typical values for free
T4 (f T4) and free T3 (f T3) fractions in mammalian plasma are 0.020.05
and 0.20.5% of the total T4 and T3 concentrations, respectively. In fish,
the f T4 fraction (ranging from 0.15 to 0.4% in salmonids) is generally
higher than in mammals, and, in contrast to mammals, exceed f T3
fractions (ranging from 0.1 to 0.2% in salmonids) (Eales et al., 1983; Eales
and Shostak, 1985).
In fishes, albumin is a common protein in plasma which can bind T4
(Richardson et al., 1994), but much less is known about other thyroid
hormone binding proteins. Cyr and Eales (1992) suggested that changes in
plasma free T4 concentrations in estradiol-treated rainbow trout were
mediated through lipoproteins and vitellogenin. Their observations were
confirmed by experimental results obtained on rainbow trout plasma
lipoproteins (Babin, 1992) and vitellogenin in killifish (Fundulus
heteroclitus) (Monteverdi and Di Giulio, 2000) and gilthead seabream
(Sparus auratus) (Funkenstein et al., 2000). Indeed, lipoproteins are
considered to be a primitive plasma hormone transport modality
(Benvenga, 1997). Only fairly recently, a full length cDNA encoding a
TTR protein was isolated from seabream liver (Santos and Power, 1999;
Santos et al., 2002). It is biochemically distinct from TTR of higher
vertebrates, i.e., it preferentially binds T3 over T4, and it does not form
dimers with retinol-binding protein as it does in mammals (Santos and
Power, 1999; Folli et al., 2003). It could well be that the relatively high f T4
fraction in rainbow trout (Cyr and Eales, 1992) results from the binding
properties of plasma proteins, rather than from a high secretion rate of the
thyroid gland.
Total plasma thyroid hormone concentrations are, thus, greatly
determined by the spectrum and concentrations of proteins in the plasma,
which, in turn, are determined by physiological and pathological factors
such as nutritional state, reproduction, disease, and developmental state
(Richardson et al., 2005), and, indeed, osmoregulatory activity of fishes
(Sangiao-Alvarellos et al., 2003). It is generally assumed that target cells
can only take up the free forms of thyroid hormones. Free T4 and f T3
concentrations are, therefore, more relevant to thyroid status than are
total hormone concentrations, as it is in (human) clinical diagnostics
(Midgley, 2001). We have measured increased f T4 levels, with f T3 levels
unchanged, in gilthead seabream that were adapted to low salinity water
(Klaren et al., 2007), indicating that the free thyroid hormone level is
39
responsive to an osmotic challenge. Unfortunately, many research papers

often do not report whether total or free thyroid hormone concentrations
were measured in fish plasma. Considering the (nanomolar) hormone
concentrations reported, we assume measurements mostly represent total
thyroid hormone levels.
It has long been assumed that, due to their lipophilic nature,
iodothyronines cross the plasma membrane by diffusion only. Only
recently several carrier proteins for thyroid hormones have been proposed.
They include members of the organic anion transporter family (Abe et al.,
1998; Friesema et al., 1999; Fujiwara et al., 2001) and amino acid
transporters (Friesema et al., 2001, 2003). No piscine orthologues have
been identified to date. The thyroid hormone carriers identified thus far
display different preferences for the transport of T4 and T3 (for a review
see Jansen et al., 2005). It follows that the repertoire of carriers expressed
by a cell or tissue determines the bioactivity of T4 and T3, and, hence, in
vivo or in vitro treatments with thyroid hormones do not necessarily have
to result in an intracellular hyperthyroidism.
PERIPHERAL METABOLISM
Once secreted into the circulation, thyroid hormones are subject to a
series of metabolizing pathways, which lead to major and minor
iodothyronine metabolites (Fig. 2.1). Acknowledgement of these is not
trivial, since they possess highly different reactivities towards metabolizing
enzymes and receptors. The extensive peripheral metabolism of thyroid
hormones bears an analogy to the complex posttranslational processing
seen for some peptide hormones.
Metabolic pathways other than deiodination (which is treated in more
detail in the next section) involve sulfation (catalyzed by sulfotransferases)
and glucuronidation (UDP-glucuronyltransferases) to yield conjugated
thyroid hormones in mammals (Visser, 1996) and teleosts (Sinclair and
Eales, 1972; Finnson and Eales, 1996, 1997, 1998). Conjugated
iodothyronines are considered to be biologically inactive, and the
increased water solubility to facilitate urinary and biliary excretion. The
presence of glucuronidated and sulfated iodothyronines in fish bile and
urine (Parry et al., 1994; Finnson and Eales, 1996) corroborates the role of
hepatic conjugation as a clearance pathway. Interestingly, in healthy,
fasted Mozambique tilapia a substantial fraction (ca. 8%) of the total
plasma T3 pool was found to be glucuronidated (DiStefano et al., 1998).
40
Fish Osmoregulation
Fig. 2.1 Pathways of thyroid hormone metabolism (adapted from Khrle et al., 1987).
Here, T4 is chosen as the central metabolite, but most reactions are applicable to,
respectively, T3 and T2s as well. Note: T4 sulfate is not susceptible to deconjugation by
sulfatase activity (as indicated by the dashed arrow) or outer ring deiodination by D1, but
T3 sulfate is. Abbreviations: DIT, diiodotyrosine; Tetrac, tetraiodoacetic acid; Tetram,
tetraiodothyronine; Triac, triiodotetraacetic acid.
This and other observations in mammals (van der Heide et al., 2002, 2004)
hint at a role of thyroid hormone conjugation other than the facilitation
of excretion. Indeed, sulfation and glucuronidation greatly affect the
reactivity of iodothyronines towards deiodinases, receptors, binding
proteins, and cellular uptake. (Hays and Hsu, 1988; Hays and Cavalieri,
1992; Visser, 1994, 1996; van der Heide et al., 2007). When we transferred
gilthead seabream from seawater to low salinity water (1 ppt salinity), we
not only measured increased plasma f T4 levels and decreased branchial
outer ring deiodination activities, but also differential responses of enzyme
activities putatively involved in the conjugation and deconjugating
pathways of peripheral thyroid hormone metabolism (Klaren et al., 2007).
The total potential effect of secreted T4, of which the thyroid is the
only source, is very likely to be much more than the added effects of T3
and T4. Iodothyronine metabolites could well play subtle but important
roleslocally and systemicallyin organismal physiology.
41
Deiodination
Deiodination involves the enzymatic removal of an iodine atom from the
outer (phenolic) ring and/or inner (tyrosyl) ring of the iodothyronine
molecule. Outer ring deiodination of T4 is required to yield the potent
bioactive hormone 3,5,3-triiodothyronine (T3). Three mammalian
iodothyronine deiodinases (D1, D2, D3) have been characterized, and all
three are selenoenzymes with a selenocysteine in the catalytic centre, a
specific iodothyronine substrate affinity and tissue distribution, and
preference for inner or outer ring deiodination (Fig. 2.2). Only the
mammalian D1 isozyme is sensitive to inhibition by the thyrostatic PTU
(see reviews: Khrle, 1999; Bianco et al., 2002; Kuiper et al., 2005).
Teleost deiodinases resemble their mammalian counterparts in their
primary structure, but, although it has been suggested that they are more
Fig. 2.2
Pathways for inner and outer ring deiodination.
42
Fish Osmoregulation
similar to human orthologues than is generally accepted (Mol et al., 1998),

some peculiar biochemical differences certainly exist (see review: Orozco
and Valverde-R, 2005). Teleost D1 is relatively insensitive to PTU
inhibition (Sanders et al., 1997; Orozco et al., 2003; Klaren et al., 2005b),
as opposed to mammalian D1. In a number of teleost species (e.g., gilthead
seabream, Senegalese sole (Solea senegalensis)), D1 activity is inhibited by
dithiothreitol, the thiol cofactor that, in vitro, potently activates
mammalian deiodinases (Mol, 1996; Klaren et al., 2005b). Moreover,
whereas the inner ring deiodination rates of sulfated T4 and T3 by rat D1
is greatly enhanced over that of native iodothyronines, no evidence was
found for the deiodination of thyroid hormone sulfates in rainbow trout
liver (Finnson et al., 1999).
Deiodinases are sensitive to the thyroid status of an animal, i.e., they
are regulated by the very substrates and products that these enzymes use
and produce. Indeed, Eales and colleagues (Fok and Eales, 1984; Eales and
Finnson, 1991) found that outer ring deiodination of T4 in the liver of
rainbow trout was reduced upon treatment with T4 or T3. Evidence for
the direct involvement of deiodinases was obtained in Nile tilapia, where
hepatic D1 and D2 mRNA levels and deiodinating activities were
upregulated, but D3 activities in brain, gills and liver were reduced upon
methimazole-induced hypothyroidism (Mol et al., 1999; Van der Geyten et
al., 2001). The highest D2 activities are detected in the teleost liver, and
experimental hyperthyroidism decreases, and hypothyroidism increases
hepatic D2 gene expression and enzyme activity (Mol et al., 1999; Van der
Geyten et al., 2001; Garca et al., 2004). Hepatic and renal D1 enzyme
activities appear to be insensitive to experimental hyperthyroidism,
induced by treatment with T3, in several teleost species (Finnson and
Eales, 1999; Mol et al., 1999; Garca et al., 2004), although in killifish
hepatic D1 mRNA levels were reduced upon 12-24 h treatment with T4,
T3, or, surprisingly, 3,5-T2 (Garca et al., 2004). Interestingly, the choice
of treatment to induce hyperthyroidism is relevant here. Van der Geyten
et al. (2005) have recently shown that treatment of Nile tilapia with T3
does not affect hepatic and renal D1 activities, in accordance with the
results mentioned above, whereas treatment with T4 increased hepatic D1
activity. Not only because of the differential regulation of deiodinases by
thyroid hormone, but also as a result of the specific preferences for the
transport of thyroid hormones by carrier proteins (see previous chapter),
experimental treatments with T4 or T3 are clearly not equivalent, and will
43
result in physiologically different hyperthyroid states. This should be an

important consideration in the design of physiological experiments.
D1 and D2 possess outer ring deiodination activities, and are thus
involved in the activation of the thyroid prohormone T4 to T3. On the
contrary, D3 possesses an inner ring deiodination activity only, and its
obvious role in thyroid hormone metabolism is the irreversible inactivation
of T4 and T3. It should be appreciated that the regulation of deiodinases
by iodothyronines can confound experimental results obtained on fishes
with experimentally induced hyper- or hypothyroidism. Experimental
treatments are mostly aimed at attaining elevated or decreased systemic
plasma thyroid hormone levels, but hyper- or hypothyroidism thus induced
is not always reflected locally, at the organ or cellular level. Indeed,
experimentally elevated plasma or tissue T4 levels in fish do not always
result in elevated T3 concentrations (Blaschuk et al., 1982; Fok and Eales,
1984; Inui et al., 1989; Peter et al., 2000). Deiodinases are important
determinants of the thyroid status of an animal, and could be useful
parameters in assessing the activity of the thyroid peripheral system.
Indeed, in rainbow trout and gilthead seabream, osmotic challenge
resulted in altered outer ring deiodination activities in gills, liver and
kidney (Orozco et al., 2002; Klaren et al., 2007).
THYROID HORMONE TARGETS
Although (non-genomic) actions of T4 and T3 have been described in
mammalian cells (see review: Davis et al., 2002), T4 is still traditionally
considered to be a prohormone with few or no biological activities. The
most biologically potent iodothyronine, T3, binds intracellularly to a
thyroid hormone receptor (TR), a type-2 nuclear receptor of which four
isoforms (a1, a2, b1, b2) are currently known. The T3-TR ligandreceptor complex heterodimerizes with a retinoic X receptor (RXR), and
can then bind to a thyroid response element (TRE) in or near the
promoter region of thyroid hormone responsive genes, leading to the
activation or suppression of gene expression (see review: Yen, 2001).
Thyroid Hormone and Na,K-ATPase
The actions of T3 are truly pleiotropic, as exemplified by DNA-microarray
analyses of hepatic gene expression in hyper- and hypothyroid mice (Feng
et al., 2000). From a fish osmoregulatory perspective, however, the most
44
Fish Osmoregulation
important target of T3 action is likely to be the Na,K-ATPase sodium

pump. The Na,K-ATPase holoprotein is a tetramer consisting of two aand two b-subunits. In mammals, both subunits are sensitive to T3 in an
organ-specific manner (Horowitz et al., 1990; Bajpai and Chaudhury,
1999; Yalcin et al., 1999). Several studies point to the regulatory actions
of T3 treatment on in vitro teleost branchial, renal and hepatic Na,KATPase activities (De et al., 1987; Peter et al., 2000; Shameena et al.,
2000), indicating that in fishes (subunits of) the sodium pump also are
sensitive to T3. Na,K-ATPase is a pivotal enzyme involved in ion transport
and osmoregulation, and its activity in branchial and renal epithelia is
increased upon seawater acclimation of fish (Madsen et al., 1995; Seidelin
et al., 2000). This would make thyroid hormones a priori important
determinants of osmoregulatory capacity in teleosts.
Considering the presence of positive thyroid response elements in the
Na,K-ATPase subunit gene promoter regions, one would predict increased
branchial and renal enzyme activities upon thyroid hormone treatment.
However, evidence for this relation is still inconclusive. Indeed, in
cultured pavement cells from rainbow trout treatment with T3 resulted in
a ca. 35% increase in Na,K-ATPase activity (Kelly and Wood, 2001).
However, this effect was not T3-dose dependent and was not correlated
with net transepithelial Na+ fluxes. In in vivo studies in salmonids, plasma
T4 concentrations were found to be correlated with branchial Na,KATPase activity (measured in vitro as the rate of ouabain-sensitive ATP
hydrolysis) (Folmar and Dickhoff, 1979; Virtanen and Soivio, 1985).
Contrary, Na,K-ATPase activities were unaffected (Saunders et al., 1985;
Dang, 1986; Madsen, 1989, 1990; Shrimpton and McCormick, 1998;
Mancera and McCormick, 1999) or even reduced (Omeljaniuk and Eales,
1986) by treatment with T4 or T3 in a number of teleost species. Peter
et al. (2000) measured a tissue-specific response of Na,K-ATPase to
treatments with T4 or T3 in freshwater Mozambique tilapia; at low doses
branchial sodium pump activities were increased by 50 to 70%, whereas
those in kidney were decreased. At higher hormone doses, these effects
disappeared. We have as yet found no satisfying explanation for this result,
and, indeed, for the lack of effect of thyroid hormone treatment on in vitro
Na,K-ATPase activities. We have to consider the methodology of assaying
Na,K-ATPase activities in vitro, which is mostly performed on
homogenates or partially purified membrane preparations. This does not
allow a discrimination between enzyme activities in (sub)cellular fractions
involved in osmoregulatory processes (i.e., branchial chloride cells,
45
basolateral membranes) and in fractions that are not (i.e., branchial

pavement cells, Na,K-ATPase in intracellular compartments). The
measurement of [3H]-ouabain binding sites (Clausen, 1996) in teleost
branchial and renal tissues could be very fruitful to assess the effects of
thyroid hormones on Na,K-ATPase.
THYROID GLAND REGULATION
Pituitary GlandTSH
Thyroid-stimulating hormone (TSH, or thyrotropin) is the key regulating
factor of thyroid function. Treatment with heterologous TSH results in
elevated plasma T4 levels in a number of teleost species in vivo (Grau and
Stetson, 1977; Brown and Stetson, 1983; Specker and Richman, 1984;
Brown et al., 1985; Leatherland, 1987; Inui et al., 1989; Byamungu et al.,
1990; Bandyopadhyay and Bhattacharya, 1993) and an increased release
of T4 from Hawaiian parrotfish (Scarus dubius) thyroid in vitro (Grau et al.,
1986; Swanson et al., 1988). The parrotfish thyroid, which consists of two
distinct lobesand this is an exceptional organization in fish, as the
thyroid gland is diffusely organised in other speciesis particularly
suitable for static or perifusion incubations. In vitro studies conducted on
parrotfish performed by Grau et al. (1986) and Swanson et al. (1988) are
unique and have allowed a direct assessment of thyroid gland output and
other aspects of thyroid gland physiology. Still, with the help of detailed
anatomical knowledge, careful dissection and perhaps a mild digestion of
surrounding tissues, it could well be feasible to subject thyroid glands from
other teleost species to in-vitro examinations, as was performed by
Bhattacharya et al. (1976a). In this respect, the technique developed by
Toda et al. (2002) to maintain cultured porcine thyroid follicles in a threedimensional extracellular matrix environment could be promising.
A negative feedback exists between plasma thyroid hormones and
TSH secretion by pituitary thyrotrophs. Specifically, in several teleost
species T4 and T3 down-regulate the pituitary TSH b-subunit mRNA
content (Larsen et al., 1997; Pradet-Balade et al., 1997, 1999; Sohn et al.,
1999; Yoshiura et al., 1999; Chatterjee et al., 2001; Geven et al., 2006).
HypothalamusTRH and CRH
Thyrotropin-releasing hormone (TRH), a hypothalamic tripeptide (pGluHis-Pro-NH2) controls pituitary TSH cells and also functions as a
46
Fish Osmoregulation
neurotransmitter throughout the central nervous system. It has a

widespread distribution in the teleost brain (Kreider et al., 1988; Batten
et al., 1990a, b; Hamano et al., 1990; Matz and Takahashi, 1994; Matz and
Hofeldt, 1999; Diaz et al., 2001, 2002). In the brain and pituitary gland of
white sucker (Catostomus commersonii) and salmonids two TRH-R
subtypes have been identified (Schwartzentruber and Omeljaniuk, 1995;
Ohide et al., 1996; Harder et al., 2001; Kumar and Trant, 2001). Although
TRH circulates peripherally in Mozambique tilapia (Lamers et al., 1994),
no TRH-binding sites outside the fish brain are known (for reviews see
Burt and Ajah, 1984; Kumar and Trant, 2001).
Recently, it has been shown that TRH upregulates TSH b-subunit
gene expression in a teleost (bighead carp, Aristichtys nobilis) pituitary
(Chatterjee et al., 2001). However, the hypophysiotropic and thyrotropic
effects of TRH differ greatly among species. In arctic charr (Salvelinus
alpinus) low doses (0.1 mg/g body weight) of TRH elevate plasma T4
levels, but rainbow trout responded only to higher doses (1 mg/g) (Eales
and Himick, 1988). In many other fishes, TRH has been reported to be
without effect on TSH release from pituitary cells or on the thyroid status
of the animal, whereas the release of growth hormone, prolactin or aMSH
was stimulated (Gorbman and Hyder, 1973; Dickhoff et al., 1978; Lamers
et al., 1994; Melamed et al., 1995; Kagabu et al., 1998; Larsen et al., 1998).
TRH clearly is a misnomer for the piscine tripeptide pGlu-His-ProNH2.
Corticotropin-releasing hormone (CRH) is abundantly expressed in
the teleost brain (Pepels et al., 2002a). Although in Mozambique tilapia
around 80% of the total brain immunoreactive CRH content is localized
outside the hypothalamus (Pepels et al., 2002b), the presence of CRH in
neurons of the hypothalamic preoptic area (nucleus preopticus, NPO),
which project to the pituitary, is of special relevance for pituitary gland
regulation (see review: Meek and Nieuwenhuys, 1998). The classical
action of hypothalamic CRH in all vertebrates including teleosts is the
regulation of the release of adrenocorticotropic hormone (ACTH) from
the pituitary pars distalis which, in turn, regulates the secretion of cortisol
from the head kidneys interrenal cells during a stress response (Ando
et al., 1999; Huising et al., 2004; Metz et al., 2004; Flik et al., 2005). The
involvement of CRH in the regulation of the thyroid gland can be inferred
from the observation that lesions of the NPO in tilapia resulted, after
10 days, in increases of plasma T4 and rT3, but not T3 (Sukumar et al.,
1997). Lesions in other parts of the hypothalamus, i.e., the anterior and
47
posterior nucleus lateralis tuberalis, had no effect, and the pituitary

contents of growth hormone and prolactin remained unchanged by any of
these lesions (Sukumar et al., 1997), indicating a specific thyrotropic
action of a specific cell population in the NPO. Reversely, treatment of the
catfish Clarius batrachus with thyrostatics resulted in a decrease of nuclear
dimensions of NPO cells (Dixit, 1976). Heterologous CRH and other
members of the CRH family, e.g., urotensin I and sauvagine, were found
to be potent stimulators of TSH release from cultured pituitary cells from
coho salmon (Larsen et al., 1998). Intracerebroventricular administration
of ovine CRH (oCRH) in fed goldfish (Carassius auratus) decreased total
thyroid T4 and T3 content. In fasted goldfish, oCRH treatment increased
the free T4 and decreased free T3 contents of the thyroid (De Pedro et al.,
1995).
It appears that through an involvement of the shared signal molecule
CRH, the corticotrope and thyrotrope axes in fishes are intertwined [(see
Khn et al. (1998) for other vertebrates)], and this would open an
interesting field of research. We have hypothesized that thyroid hormones,
the release of which is regulated by CRH, feed back on the NPO to
modulate the CRH activity which would have a concomitant effect on the
hypothalamus-pituitary-interrenal axis. Interesting results were obtained
from studies where T4 treatment or thyroidectomy of rats resulted in
reciprocal changes in the expression of CRH mRNA and other
hypothalamic peptides as well in the paraventricular nucleus (homologous
to the teleost NPO) (Ceccatelli et al., 1992; Dakine et al., 2000). We have
also found that carp with experimentally induced hyperthyroidism
displayed a marked hypocortisolinemia, and this correlated with increased
mRNA-levels of CRH binding protein, a primitive endogenous CRH
antagonist (Huising and Flik, 2005; Geven et al., 2005). We interpret this
as a proof of principle of the hypothesis that thyroid hormones affect the
activity of the hypothalamus-pituitary-interrenal axis at a central,
hypothalamic level. This could also form the molecular basis of the
interactions between thyroid hormones and cortisol in fish. It has been
observed that cortisol treatment reduces plasma T4 in European eel
(Anguilla anguilla) and coho salmon (Redding et al., 1984, 1986), but
negative effects have also been observed (Leatherland, 1987; Redding
et al., 1991). Direct proof for the synergistic effect of thyroid hormone and
cortisol on osmoregulatory capacity comes from cultured pavement cell
epithelia from rainbow trout where, compared with T3 alone, a
48
Fish Osmoregulation
co-incubation of T3 and cortisol decreased unidirectional Na+- and Clfluxes under symmetrical culture conditions (Kelly and Wood, 2001).
Hypothalamic actions of thyroid hormones could well be involved in
the synergistic or additive effects of thyroid hormones and cortisol in
teleost osmoregulation (for review see McCormick, 2001).
OSMOREGULATORY ASPECTS OF THYROID HORMONES
Ontogeny and Development of Osmoregulatory Capacity
Early experiments by the thyroidology pioneer J. Friedrich Gudernatsch
clearly indicated the determining role of the thyroid gland in (amphibian)
metamorphosis. The involvement of the thyroid in teleost metamorphosis
and ontogeny is exemplified by the observation that exogenous
administration of T4 induced metamorphosis in flatfish whereas the
thyrostatic thiourea inhibited it (Solbakken et al., 1999; Stickney and Liu,
1999). Reversely, T4 rescued zebrafish (Danio rerio) from developmental
arrest induced by thyrostatics (Brown, 1997). Thyroid hormones are
involved in many ontogenetic events, e.g., the development of the
olfactory region of the brain, and of ultraviolet photosensitivity of the
retina during parr-smolt transformation of salmonids (Browman and
Hawryshyn, 1994; Morin et al., 1997; Alexander et al., 1998). The
pervasive effects of thyroid hormones on ontogenetic development are
perhaps best illustrated in the teleost ice goby (Leucopsarion petersii) in
which an inactive thyroid gland is considered to be causal to the neotenic
phenotype of the adult animal (Harada et al., 2003). The developmental
effects of thyroid hormones are regulated through the differential
expression of thyroid hormone receptor subtypes, which are expressed
already before midblastula stage in zebrafish embryos (Liu et al., 2000) and
which are temporally and regionally differentially expressed in several
teleost species (Yamano and Miwa, 1998; Power et al., 2001; Marchand
et al., 2004).
Anadromic and catadromic species, in particular, encounter greatly
varying environments during their natural life span. Plasma thyroid
hormone levels vary with the migration of salmonids (Eales, 1965; Sower
and Schreck, 1982; Cyr et al., 1988; Youngson and Webb, 1993; Iwata,
1995; Persson et al., 1998), indicating that they are possibly responsive to
environmental salinity. However, it cannot be excluded that the observed
changes in plasma thyroid levels are involved in the adaptation to other
49
environmental factors than salinity, or aspects other than strictly

osmoregulation, e.g., parr-smolt transformation and spawning. More direct
evidence for the involvement of thyroid hormones in osmoregulation
comes from studies where fishes, ceteris paribus, were transferred to
different salinities and where changes in thyroid gland morphology, and
plasma levels of T4 and/or T3 were measured (Olivereau et al., 1977;
Folmar and Dickhoff, 1979; Redding et al., 1984, 1991; Grau et al., 1985;
Klaren et al., 2007). Yet, in this context, there have been reports of
negative results, i.e., no changes in thyroid hormone plasma levels
(McCormick and Saunders, 1990; Redding et al., 1991).
Treatment of animals with the thyrostatic thiourea apparently reduces
their osmoregulatory capacity (Knoeppel et al., 1982; Madsen, 1989;
Schreiber and Specker, 1999). Results from these studies are equivocal,
e.g., the treatment with T4 not always rescues from the treatment with the
thyrostatic, indicating that the actions of thiourea in fish are not always
specifically targeted at the thyroid gland.
HETEROTOPIC THYROID FOLLICLES IN
OSMOREGULATORY ORGANS
In contrast to the compact thyroid gland found in higher vertebrates, in
virtually all teleosts and adult Cyclostomata the thyroid gland consists of
rather diffusely scattered follicles (single or in small groups) in the region
ventral to the pharynx, along the ventral aorta and where the branchial
arteries branch off. Almost a century ago, Gudernatsch (1911) reported on
the dispersal of thyroid follicles over larger areas in the subpharyngeal
region. His early observations coincided with reports on so-called thyroid
carcinomas in brook trout (Salvelinus fontinalis) (Marine and Lenhart,
1910, 1911). Baker-Cohen (1959), who summarized the pertinent
literature on extrapharyngeal thyroid tissue, concluded that in several
teleost species the kidney was the most common location of heterotopic
thyroid follicles. Ever since, reports have appeared on heterotopic thyroid
follicles mostly in the head-kidney (pronephros) and trunk-kidney
(opistonephros) area in a large number of species, with a surge of
publications some three decades ago (Chavin, 1956; Olivereau, 1960;
Lysak, 1964; Frisn and Frisn, 1967; Srivastava and Sathyanesan, 1967,
1971a, b; Peter, 1970; Qureshi, 1975; Bhattacharya et al., 1976a, b; Joshi
and Sathyanesan, 1976; Qureshi and Sultan, 1976; Qureshi et al., 1978;
Agrawala and Dixit, 1979; Sharma and Kumar, 1982). (See Figure 2.3 with
50
Fish Osmoregulation
Fig. 2.3 Thyroid follicles in tissues of common carp (Cyprinus carpio) treated with the
trichrome Light Green-Orange G-fuchsin which stains the follicular colloid bright red.
a. Subpharyngeal region where thyroid follicles are arranged around the ventral aorta.
b. Head-kidney tissue. c. Trunk-kidney tissue.
our unpublished results on common carp, Cyprinus carpio.) Despite the

obvious involvement of head-kidney and kidney in fish osmoregulation,
the subject of renal heterotopic thyroid follicles has received little
attention since.
The head-kidney, an organ analogous to the adrenal glands in
mammals, is unique to teleost fish. Chromaffin cells and interrenal cells
produce and secrete catecholamines and cortisol, respectively.
Heterotopic thyroid follicles, when present, are functional as evidenced by
the accumulation of iodine and synthesis of the thyroxine precursors MIT
and DIT, and T4, processes that are sensitive to TSH and thyrostatics. In
some species head-kidney iodine accumulation exceeds that in the
pharyngeal region (our unpublished results on common carp, Cyprinus
carpio) and shows a seasonal variation (Chavin and Bouwman, 1965;
Srivastava and Sathyanesan, 1971a; Bhattacharya et al., 1976a). Thus,
heterotopic thyroid follicles must be active endocrine tissues, sensitive to
physiological regulators. It has been suggested that development of
heterotopic thyroid tissue may reflect a compensatory mechanism to
iodine deficiency, as iodide supplementation prevented but not reversed
the development of heterotopic thyroid tissue (Baker-Cohen, 1959).
However, ca. 40% of the cases of renal heterotopic thyroid reviewed by
Baker-Cohen were animals with normal, non-goitrous pharyngeal thyroid
tissue that probably did not experience an iodine deficiency. It may be that
heterotopic follicles concern metastatic thyroid carcinoma, as teleostean
thyroid tissue appears particularly sensitive to carcinogens and highenergy radiation (Woodhead and Scully, 1977; Blasiola et al., 1981;
Hoover, 1984; Woodhead et al., 1984; Bunton and Wolfe, 1996; Chen
et al., 1996; Toussaint et al., 1999). Indeed, a major part of Baker-Cohens
51
observations were performed on a platyfish strain (BH strain) which has a

very high thyroid tumour incidence compared to other strains. Moreover,
heterotopic renal thyroid follicles occur not only in feral fish, but also in
fish kept or bred under laboratory conditions (Baker-Cohen, 1959; Frisn
and Frisn, 1967; Qureshi, 1975). Interestingly, Baker-Cohen (1959)
observed normal thyroid tissue in head-kidney of platyfish of strain 30, a
strain that does not develop thyroid tumours while the BH strain does. We
observed thyroid follicles in head-kidney and renal tissue of our laboratorybred and -raised carp and tilapia (unpublished results). The presence of
heterotopic thyroid follicles is most likely a normal anatomical feature in
healthy animals. It is interesting to note that in fish the location of the
majority of thyroid follicles, i.e., near the branchial efferents of the ventral
aorta, kidney and head-kidney, always is in a well-innervated area and
associated with a portal system. This could have a physiological
significance for systemic thyroid hormone homeostasis.
The preference of heterotopic thyroid follicles for residence in headkidney tissue, the close juxtaposition of iodothyronine-producing cells
with interrenal (cortisol-producing) cells, chromaffin (catecholamineproducing) cells, and haematopoietic cells strongly hints at some paracrine
relationship between thyroid and interrenal tissue. Initial experiments in
our department failed to establish an effect of T4 or T3 on the in vitro
cortisol release from tilapia head-kidney. However, the putative relation
between the head-kidney and its thyroid tissue could well be more
intricate, and deserves further and more detailed investigation.
CONCLUSION
The best known in vivo effect of thyroid hormones is the stimulation of the
basal metabolic rate, and from this alone the involvement of thyroid
hormones in osmoregulatory processes can be inferred. The multiple
effects of thyroid hormone and probably its metabolites as well on, e.g.,
development (of osmoregulatory capacity, sensu stricto) and Na,K-ATPase
activities strengthen the important role of iodothyronines in teleost
osmoregulation. However, a generalized mode of action does not, as yet,
emerge, not only because of the relative paucity in physiological
experimentation in teleost thyroidology, but also because of the
synergistic/additive effects of thyroid hormones with cortisol (and growth
hormone) in osmoregulation.
Fish Osmoregulation
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Fish Osmoregulation
+0)26-4
!
Diet and Osmoregulation
Francesca W. Ferreira1 and Bernardo Baldisserotto2, *
INTRODUCTION
Fish adapted to freshwater and waters with low salinity present a diffusive
ion loss to the environment via gills and skin, as well as by feces and urine.
This ion loss must be compensated by an active influx from the water by
the gills (Wood, 2001), from the diet by the intestine (Dabrowski et al.,
1986; Buddington and Diamond, 1987; Bog et al., 1988; Baldisserotto
et al., 1993; Kerstetter and White, 1994; Baldisserotto and Mimura, 1995;
Bijvelds et al., 1998), and in some species as the swamp eel, Synbranchus
marmoratus, it might be also complemented by the skin (Stiffler et al.,
1986). Another complicating factor in freshwater fishes is that most
studies of in vitro intestinal absorption/transporters were done with fasting
fishes. Feeding drastically changes the ionic situation of rainbow trout,
Oncorhynchus mykiss, intestine (Dabrowski et al., 1986), and the addition
Authors addresses: 1Departamento de Biologia e Qumica, Universidade Regional do Noroeste
do Rio Grande do Sul, 98700.000 Iju, RS, Brazil.
2
Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria,
97105.900 Santa Maria, RS, Brazil.
*Corresponding author: E-mail: bernardo@smail.ufsm.br
68
Fish Osmoregulation
of several amino acids or glucose to the mucosal side of the medium

bathing to intestines in vitro increases the flow of Na+ toward the serosal
side in various teleost species (Ferraris and Ahearn, 1984; Vilella et al.,
1988, 1989; Bog and Pres, 1990). In addition, intestinal Ca2+ absorption
is affected by the diet (Baldisserotto et al., 2006).
The drinking rate of freshwater teleosts is low (370-1400 mlh1kg1)
(see Flik et al., 1985), but the intestine (or the pyloric ceca, when present)
can absorb Na+, Cl, Ca2+, and Mg2+ (and probably other ions) provided
by feeding (Dabrowski et al., 1986; Buddington and Diamond, 1987; Bog
et al., 1988; Baldisserotto et al., 1993; Kerstetter and White, 1994;
Baldisserotto and Mimura, 1995; Bijvelds et al., 1998). Therefore, diet can
be an important ion source for osmoregulatory needs of fish living in
hyposaline environments. Dietary salt supplementation can also decrease
energy spent on osmoregulation and consequently more will be available
for growth (Gatlin et al., 1992; DCruz and Wood, 1998).
On the other hand, fish that live in waters with high salinity have
problems of excessive ion influx, which must be eliminated by the gills and
urine. The digestive tract absorbs ions, but the aim of this process is to
provide absorption of the ingested water and not ions from the diet (Kirsch
et al., 1984). Therefore, present chapter will deal mainly with the
contribution of the diet to osmoregulation of fish adapted to low salinities.
Moreover, emphasis will be on the direct effects of dietary composition on
osmoregulation and not indirect effects (morphological changes) due to
lack of specific nutrients as vitamins, for example.
DIETARY Na+ AND Cl
Rainbow trout can survive for long fasting periods without a significant
decrease on blood Na+ concentration (Heming and Paleczny, 1987).
Consequently, Na+ branchial influx is adequate for maintaining ionic
balance in fish even when food consumption (and, consequently, dietary
Na+ intake) is low, as in winter (Smith et al., 1989). However, intestinal
influx of dietary Na+ in rainbow trout collected from the wild in summer
is similar to branchial influx (Smith et al., 1989). Apparently, most dietary
Na+ ingested is absorbed by the gut (Salman, 1987; Smith et al., 1995),
because feces have a low amount of salts even in rainbow trout fed with
high salt content in the diet (Salman and Eddy, 1988). Rainbow trout fed
high NaCl diets (1.8 and 3% Na+) showed a decrease of 40.8 and 44.0%
on waterborne Na+ whole body uptake rates relative to controls (diet with
69
0.6% Na+). Moreover, Na+ efflux was 12% and 38% higher in fish fed
1.8% and 3% sodium-enriched diets, respectively. The increase of plasma
Na+ concentration due to high dietary Na+ (38.1% in fish fed with 3%
sodium-enriched diet) (Fig. 3.1) probably causes a downregulation of a
branchial uptake route through an apical sodium channel, which reduces
waterborne Na+ uptake. Fish fed high-sodium diets (3%) also drank 58%
more water than controls (Pyle et al., 2003). This increase on drinking rate
is needed to counterbalance the increase on plasma Na+ concentration
(Salman and Eddy, 1988).
An increase of dietary NaCl from 2 to 12% in rainbow trout promoted
a two-fold increase of the number of chloride cells in the gills and gill Na+/
K+- ATPase activity (Salman and Eddy, 1987). Na+/K+- ATPase activity
in the proximal intestine (pyloric ceca and anterior intestine) was also
stimulated by Na+-supplemented diets in rainbow trout (Pyle et al., 2003),
but not in bluegill, Lepomis macrochirus (Musselman et al., 1995). The
proportion of chloride cells related to total branchial cells also increased
from 8% in rainbow trout fed with 1.3% dietary salt, to 10.5% in fish fed
with 12% dietary NaCl (Salman and Eddy, 1987). High dietary NaCl
(11.6%) did not alter the typical freshwater renal mechanism in rainbow
Fig. 3.1 Total Na+ levels in gut tissue and plasma of rainbow trout fed for 7 days with
different Na+ levels in the diet (and also exposed to 20 mg L1 waterborne Cu for 6 h).
Adapted from Pyle et al. (2003).
70
Fish Osmoregulation
trout, where the majority of filtered ions are reabsorbed to produce a

relatively large volume of dilute urine. However, there was an increase on
glomerular filtration rate and urinary flow rate, which approximately
doubled the Na+ and Cl urinary excretion rate. Even with this increase
of salt urinary excretion rate, salt renal excretion represented only 10% of
the total body loss and is similar to fecal salt loss (Salman and Eddy, 1988).
Increase of drinking rate (Pyle et al., 2003), gill water permeability and
intestinal water absorption would provide the water needed for this
increase of urinary flow rate (Salman and Eddy, 1988).
The principal mechanism for Na+ homeostasis is the variation on
branchial Na+ influx and efflux rates, and the main way of excreting large
salt loads is to increase Na+ branchial efflux (Smith et al., 1995; Pyle et al.,
2003). In rainbow trout fed on freshwater shrimp Gammarus pulex 60% of
the ingested Na+ was absorbed within 5 h (Smith et al., 1989). Similarly,
rainbow trout receiving a commercial diet supplemented with 12% NaCl
constituted a mean Na+ load of 36.2 mmolkg fish1, of which around 85%
was absorbed within 7 h (maximum time of the experiment). Absorption
from the gut increased the Na+ plasma levels when compared with levels
in unfed fish, and within 1 h, branchial Na+ efflux increased and remained
high for 7 h, indicating that excretion of excess Na+ was incomplete at the
end of this period (Smith et al., 1995). Blood Cl levels were unchanged
in brook trout (Salvelinus fontinalis) fed a NaCl load of 15.3 mmolkg fish1,
but ingestion of 46 mmol.kg fish1 increased blood Cl levels up to 40%
above control values 7 h after feeding. Blood Cl levels returned to control
values after 24 h, but ingestion of higher NaCl load (77 mmolkg fish1) led
to a prolonged increase in blood Cl- levels and in many cases, death
(Phillips, 1944). Plasma Cl levels in bluegill maintained in freshwater and
fed diet supplemented with 2 or 4% NaCl were also higher than in fish kept
in freshwater and fed a diet without NaCl supplementation (Musselman
et al., 1995).
In acidic water, excess H+ can inhibit the Na+/H+ exchanger (Potts,
1994) and create a gradient too steep for further extrusion of protons (Lin
and Randall, 1991), reducing Na+ uptake by the gills. Moreover, high H+
concentrations disrupt the tight junctions of gill epithelia, increasing ion
loss by a paracellular route, leading to whole body ion loss, as observed in
rainbow trout (McDonald and Wood, 1981) and silver catfish, Rhamdia
quelen (Zaions and Baldisserotto, 2000). Under these conditions, dietary
salts may become very important in maintaining body ion levels during
71
acid stress (DCruz and Wood, 1998). Starved fish (or fed with a very
limited diet) showed ionoregulatory changes during exposure to acidic
environment (DCruz et al., 1998), but when they were fed with adequate
amount of salts the effect of low pH was reduced or did not occur (Dockrey
et al., 1996; DCruz et al., 1998). Therefore, dietary salt can replace
branchial ion loss (DCruz and Wood, 1998).
Some studies proposed that acidic pH may impair growth in rainbow
trout due to a decrease on food consumption (for review see DCruz and
Wood, 1998), as was observed in silver catfish (Copatti et al., 2005).
However, Dockray et al. (1996), Reid et al. (1996, 1997) and DCruz et al.
(1998) verified that chronic exposure of rainbow trout to low pH seemed
to stimulate appetite. Rainbow trout exposed to acidic pH for 28 days and
starved showed significantly lower plasma Na+ (but not whole body Na+,
Cl and K+) than before the acid challenge. Those fed with a low NaCl
diet (0.10.18%), independently of energy content, also presented a
decrease on plasma Na+ and whole body Na+ and Cl (the last, only the
low energy diet), but fish fed with 0.6% NaCl did not show any ionic
imbalance. Therefore, is the salt content of the food rather than the energy
content that is critical in protecting against the effect of acidic pH (DCruz
and Wood, 1998)?
An adequate dietary Na+ level could have lower metabolic cost when
associated with active branchial ion transport, and the saved energy could
be used for growth (Smith et al., 1989). Atlantic salmon (Salmo salar) has
a whole body Na+ content of 25 mmol/kg (Talbot et al., 1986), so Smith
et al. (1989) estimated that a 10 g fish (whole body Na+ content of 0.25
mmol) doubling in weight over a year would need 0.25 mmol Na+.
According to the same authors, this amount is easily obtained by feeding
and branchial uptake because total Na+ influx in rainbow trout in June is
over 1000 times greater. It must also be considered that branchial Na+
fluxes may be rapidly adjusted to diet changes (Smith et al., 1995), and
therefore, a high Na+ diet might not improve growth in rainbow trout in
optimum water conditions. However, when fish are exposed to acidic pH
branchial ion influx is lower and the efflux is higher than in neutral waters,
and dietary salt supplementation may help to maintain ionic balance
(DCruz and Wood, 1998).
Salinity has variable effects on growth of euryhaline species, and
growth is not necessarily maximal at isosmotic conditions (Brett, 1979;
Musselman et al., 1995; Likongwe et al., 1998). Red drum (Sciaenops
72
Fish Osmoregulation
ocellatus) is commonly found in waters with 2040, and juveniles

growth is improved in freshwater with a diet supplemented with 2% NaCl
or 2% NaCl + 2% KCl. However, NaCl dietary supplementation did not
affect any change in growth of juveniles exposed to brackish (6) and
seawater (35); neither was blood osmolality of fish maintained in fresh
or brackish water and transferred to seawater. These results suggest that
salinity of 6 may be close to the threshold at which dietary salt
supplementation promotes growth in red drum (Gatlin III et al., 1992).
Chinook salmon (Oncorhynchus tshawytscha) fed diets supplemented with
7% NaCl or 5% NaCl + 2% KCl showed higher tolerance to seawater
transfer (Zaugg et al., 1983). Diets supplemented with 10% NaCl also
improved survival to seawater transfer of two tilapia species (Oreochromis
mossambicus and O. spilurus) and the hybrid O. aureus O. niloticus
(Al-Amoundi, 1987), as well as brown trout (Salmo trutta) (Arzel et al.,
1993). Nile tilapia (O. niloticus) maintained in freshwater and fed diet
supplemented with 8% NaCl for 30 days showed a higher growth rate than
those fed diet without NaCl supplementation, while dietary NaCl did not
change significantly growth rate in fish kept in brackish water (10 and
20) (Fontanhas-Fernandes et al., 2002). However, Nile tilapia fed on a
diet of 8% NaCl presented significantly lower plasma Cl and osmolarity
after transference to brackish water than those fed diet without NaCl
supplementation, indicating a reduction of osmotic imbalance
(Fontanhas-Fernandes et al., 2001).
DIETARY Ca 2+
Fish take up calcium for growth and homeostasis predominantly via the
gills, directly from the water. This branchial Ca2+ uptake is an active and
a more or less continuous process and largely independent of waterborne
Ca2+ (Flik, 1996). Fish also take up Ca2+ from food or water drunk by the
intestine (Flik et al., 1993a, b; Flik and Verbost, 1995), but under normal,
no stressed conditions, the drinking rate of freshwater fish is very low, and
the contribution of the intestine to Ca2+ uptake is restricted to dietary
calcium (Flik, 1995). The contribution of gills and intestine to Ca2+
uptake is variable and depends on waterborne and dietary Ca2+
concentration. In fish exposed to low waterborne Ca2+ the relative
contribution of the food increases, whereas feeding low-Ca2+ diets
stimulates the branchial uptake. There is also evidence that fish rely on
Ca2+ intestinal uptake when extensive amounts of Ca2+ are required for
73
gonadal maturation (Flik et al., 1995). A total lack of dietary Ca2+ can be
completely compensated by branchial uptake, but very low waterborne
Ca2+ induces hypocalcemia and impairs growth (Shoenmakers et al., 1993;
Flik et al., 1996).
Low waterborne (0.125 mmol) and dietary Ca2+ reduced growth rate
in brook trout (Salvelinus fontinalis) (Rodgers, 1984), demonstrating that
a minimum Ca2+ uptake by gills and/or intestine is needed for normal fish
growth. Channel catfish (Ictalurus punctatus) reared in low waterborne
Ca2+ (< 0.25 mmol) required 4.5 mg Ca2+g1 food for normal growth and
tissue mineralization (Robinson et al., 1986), while blue tilapia
(Oreochromis aureus) reared in similar conditions fed with 7.5 mg Ca2+g1
food showed higher bone and scale Ca2+ concentration (but only higher
scale Mg2+ concentration and bone phosphorus concentration) than
those fed with diet deprived of Ca2+ (OConnell and Gatlin, 1994) (Fig.
3.2). Striped bass (Morone saxatilis) juveniles maintained at 0.68 mmol
Ca2+ presented high whole body waterborne Ca2+ uptake compared to
other teleosts, but still much lower than the rate of Ca2+ assimilation
necessary for optimum growth of this species (Grizzle et al., 1993).
However, dietary Ca2+ was dispensable for rainbow trout when waterborne
Ca2+ was above 0.6 mmol (Ogino and Takeda, 1978), and Ca2+supplemented diets from 3.6 to 11 mg Ca2+g1 food did not change growth
of this species when reared at water with 0.75 mmol Ca2+ (Barnett et al.,
1979).
Rainbow trout maintained at waterborne Ca2+ 1 mmol fed on high
dietary Ca2+ levels (60 mg Ca2+g1 food) showed 5264% lower whole
body waterborne Ca2+ uptake compared to fish fed with lower dietary
Ca2+ levels (20 mg Ca2+g1 food) (Baldisserotto et al., 2004 a, b) (Fig. 3.3).
A diet supplemented with CaCl2 to yield 30 mg Ca2+g1 food did not
change rainbow trout growth, but a higher dietary level of CaCl2 (60 mg
Ca2+g1 food) led to 21.6% mortality and decreased weight gain. The
deaths observed in the treatment with a high amount of CaCl2 probably
were due to metabolic acidosis and/or to a sharp increase on Ca2+ plasma
levels seen after the first feeding with this diet (Baldisserotto et al., 2004a).
However, in rainbow trout fed the same dietary Ca2+ levels but
supplemented with CaCO3, mortality was not observed (Baldisserotto
et al., 2004b). Therefore, supplementation with CaCO3 seems to be safer
than with CaCl2.
Fishes adapted to seawater drink water with a high Ca2+ content
(approximately 10 mmol/L), and do not decrease branchial Ca2+ uptake,
74
Fish Osmoregulation
Fig. 3.2 Calcium, Mg2+ and P concentrations in scales and bone of blue tilapia fed with
diets with different Ca2+ levels for 24 weeks. Data from OConnell and Gatlin, 1994.
*significantly different from 0 mg Ca2+ g1 food1 by ANOVA (P<0.05)
which suffices for growth and homeostasis. In addition, Ca2+ intestinal

absorption is greatly reduced when compared to freshwater fishes, and this
reduction is correlated with a decrease in the activity of Ca2+ transporters
in the enterocyte plasma membrane (Flik and Verbost, 1993).
DIETARY PHOSPHORUS
In addition to being a major constituent of structural components of
skeletal tissues, phosphorus is located in every cell of the body. It is an
75
Fig. 3.3 Ca2+ (A) whole body uptake of rainbow trout exposed to diets with different Ca2+
concentrations. Means + 1 SEM (N = 89). Adapted from Baldisserotto et al. (2004a, b).
*significantly different from 20 mg/g Ca2+ by One-way ANOVA and Tukey test (P<0.05)
important constituent of nucleic acids and cells membranes and is directly

involved in all the energy producing reactions of the cell. It plays an
important role on carbohydrate, lipid and amino acid metabolism and in
muscle and nervous tissues metabolism, as well as on various metabolic
process involving buffers body fluids. The phosphate metabolism of fish
has not been as well studied as that of Ca2+. Food is the main source of
phosphorus because phosphate concentrations are low in both freshwater
and seawater (approximately 0.02 mg L1). The uptake of phosphorus from
water has been repeatedly demonstrated (Lall, 2002). The absorption of
dietary phosphorus is affected by the level of phosphate in the blood.
Phosphorus accumulates mainly in soft tissues (heart, liver, kidney, and
blood) and, to a limited extent, in skeletal tissues (Mol et al., 1999).
Numerous studies with monogastric animals have shown that an optimum
dietary Ca2+/phosphorus ratio is important: an increase of dietary Ca2+/
phosphorus ratio interferes with phosphorus absorption, and conversely, a
high phosphorus/Ca2+ ratio may restrict Ca2+ absorption. Although the
magnitude of effect changes with species and forms of Ca2+ and
phosphorus present in the diet, such studies on Ca2+/ phosphorus ratio in
fish diet are limited (Lall, 2002).
76
Fish Osmoregulation
DIETARY Mg2+
In fish, as in all vertebrates, Mg2+ is found mineralized in bony tissues as
an ionized form (Mg2+) or complexed with proteins in all the tissues
(Bijvelds et al., 1998). The magnesium pool of bones and scales may be
used as a reserve to maintain normal Mg2+ levels in soft tissues when Mg2+
intake is low (Bijvelds et al., 1996). Of the remaining Mg2+ pool in the soft
tissues, only a small percentage is found in the extracellular fluid. The total
plasma Mg2+ concentration in most cases does not exceed 2 mmol L1, and
the ionic concentration is normally less than 1 mmol L1 (Bijvelds et al.,
1997). The ionic Mg2+ level in the cytoplasm is kept relatively low, i.e. in
the submillimolar range, typically representing less than 10% of total Mg2+
content to the cell (Bijvelds et al., 1998).
As Mg2+ plays an important role in cells, and intracellular and
extracellular Mg2+ levels are maintained within narrow limits, the
vertebrates must have developed effective mechanisms by which Mg2+ is
transported, stored and is concentration regulated. However, transport of
Mg2+ across intestinal and kidney epithelia, key process to the
understanding of Mg2+ regulation, is still poorly understood. The
mechanism of branchial Mg2+ uptake has not been demonstrated (Bijvelds
et al., 1998). Freshwater fish are threatened by diffusive losses of Mg2+
across the body surfaces because Mg2+ concentration in freshwater is
typically well below 0.5 mmol L1. These losses have to be compensated for
by Mg2+ uptake from the diet and from the water. Renal excretion must
be limited by reabsorption of filtered Mg2+ (Bijvelds et al., 1998).
The hydrolytic activity of ATPase is described as Mg2+ dependent.
ATPase affects the utilization of stored energy from ATP which is required
for excretion or intake of ions across the gill membranes against a
concentration gradient. The increased branchial ATPase activity during
smoltification has made this an indicator enzyme in monitoring the hypoosmoregulation ability in salmonids. The activity of Mg2+ATPase in renal
tissue displays positive correlation with branchial ATPase activity during
smoltification and may be involved on Mg2+ excretion. The concentration
of Mg2+ in freshwater differs widely from that in sea water, and dietary
Mg2+ fed in the freshwater phase possibly affects the regulatory ability of
Mg2+ in salmon transferred to seawater (El-Mowafi, 1997).
In freshwater fish dependent primarily on diet for their Mg2+ uptake,
optimal growth is usually achieved when dietary Mg2+ is 1520 mmol kg1.
Prolonged feeding with lower dietary Mg2+ content may lead to a decrease
77
on growth rate, Mg2+ depletion of the tissues, muscle dysfunction,

neurological disorders and high mortality (Bijvelds et al., 1996). A low
dietary Mg2+ intake induced high body Ca2+ levels in rainbow trout
(Cowey et al., 1977), tilapia (O. niloticus and O. mossambicus) (Dabrowski
et al., 1989; Bijvelds et al., 1997a) and guppy (Poecilia reticulata) (Shim and
Ng, 1988). Magnesium affects the permeability of the intestinal epithelium
to ions (Fordtran et al., 1985) and low luminal Mg2+ concentration,
therefore, increase epithelial permeability to ions, possibly stimulating
paracellular Ca2+ absorption (Karbach and Feldmeier, 1991). Minutes
perturbations to cellular Mg2+ homeostasis affects the acid-base regulation
of the cells involved in bone formation, and this may lead to
supersaturation of plasma Ca2+, resulting in spontaneous calcification
process in soft tissues (Driessens et al., 1987). In line with this hypothesis,
there was calcification of renal tissue in Mg2+-deficient rainbow trout
(Cowey et al., 1977).
It has been suggested that in Mg2+-deficient rainbow trout, the
increase in muscle Na+ content was due to a decrease in cell water content
coupled with an increase in extracellular volume (Cowey et al., 1977;
Bijvelds et al., 1998). Such changes in the mineral status and water
content of tissues are suggestive of changes in cell membrane permeability
that could cause an increased turnover. The action of Mg2+ on the fluidity
of cellular membranes may underlie this phenomenon. The permeability of
osmoregulatory epithelia may also be affected since it has been
demonstrated that external Mg2+ and Ca2+ levels influence gill
permeability to both water and ions (Wendelaar Bonga et al., 1993;
Bijvelds et al., 1996a, 1998). It is also plausible that this relationship
reflects the dependence of cellular ion transport mechanisms, such as
Na+/K+-ATPase (Rude, 1989), the Na+/K+/Cl symporters (Flatman,
1988) and cations channels (Horie et al., 1987; Dorop and Clausen, 1993;
Bijvelds, 1998) on Mg2+. Moreover, Mg2+ may influence ion movement
across cellular membranes through its action on membrane permeability
(Bijvelds et al., 1998).
Internally, Mg2+ may have similar actions on membrane permeability
and ion turnover in osmoregulatory organs such as the gills. For instance,
in Mozambique tilapia, low dietary Mg2+ caused proliferation of branchial
chloride cells (Bijvelds et al., 1996a), and a decrease in the Na+ influx
across the gills (Van der Velden et al., 1992b). Such changes are indicative
of an increased turnover of these cells in the gill epithelium. Renewal
78
Fish Osmoregulation
branchial epithelium may be a response to disturbance in ion transport

across the gills, since both the epithelial permeability to ions and water
(Wendelaar Bonga et al., 1993) and the activity of cellular ions
transporters (Flatman, 1993) are controlled by Mg2+ (Bijvelds et al.,
1997). Furthermore, in common carp (Cyprinus carpio), Mg2+ deficiency
is associated with changes in branchial ion regulation (an increase in
opercular chloride cell density and a decrease in branchial Na+/K+ATPase
activity) that coincide with an increased bone Na+ content (Van der
Velden et al., 1992a).
Freshwater fish depend on Mg2+ absorption from the intestinal tract
to meet most of their Mg2+ requirement (Van der Velden et al., 1992a, b).
It is widely recognized that the intestine is the most important route for
Mg2+ uptake in freshwater fish (Gatlin et al., 1992; Shearer and Asgard,
1992). At low dietary Mg2+ levels, absorption of this ion is highly efficient,
suggesting a regulated intestinal Mg2+ transport route. In this condition,
urinary Mg2+ excretion decreased, and this suggests that tubular
reabsorption is increased. As renal and intestinal epithelial cells maintain
a large potential difference (inside negative) across the plasma membrane,
an active extrusion mechanism is indicated, both to maintain the low
intracellular Mg2+ concentration and to allow transcellular Mg2+
transport (Bijvelds et al., 1996a).
Acknowledgements
B. Baldisserotto received a CNPq (Conselho
Desenvolvimento Tecnolgico-Brazil) research grant.
Nacional
de
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Fish Osmoregulation
+0)26-4
"
The Renin-Angiotensin Systems
of Fish and their Roles in
Osmoregulation
J. Anne Brown1, * and Neil Hazon2
The renin-angiotensin system (RAS) plays an important role in the

control of salt and water balance of most vertebrates. Our understanding
of this system, and its many actions, is most detailed in mammals (Bader
et al., 2001; Gociman et al., 2004; Navar and Nishiyama, 2004). Here, a
role in the pathogenesis of hypertension has led to pharmaceutical
targeting of the system and its receptors to manipulate vascular tone
(Croom et al., 2004). It is amongst the fish, however, where the actions of
the vertebrate (and pre-vertebrate) RAS developed, that we may perhaps
identify the fundamental (primitive) actions of the vertebrate RAS.
Research on fish has revealed both direct and indirect vasoconstrictor
actions of angiotensin (Olson, 1992; Bernier and Perry, 1999; Rankin et al.,
Authors addresses: 1School of Biosciences, University of Exeter, Exeter EX4 4PS, UK.
2
School of Biology, University of St Andrews, St Andrews KY16 8LB, UK.
E-mail: nhl@st-andrews.ac.uk
*Corresponding author: E-mail: J.A.Brown@exeter.ac.uk
86
Fish Osmoregulation
2004) similar to those initially seen when extracts of mammalian kidneys

were first injected into the vasculature resulting in angiotensin formation
in vivo. But amongst fish, the osmoregulatory actions of angiotensin also
play a significant part in dealing with some of the osmotic challenges they
face. In this chapter we will review our current knowledge of the reninangiotensin systems of fish, and our understanding of the roles of
angiotensin in their osmoregulatory processes, discussing both the
circulating RAS and the recent evidence of tissue-specific paracrine
systems.
RAS: THE BIOCHEMICAL CASCADE
The series of biochemical events that generate the physiologically active
peptides, angiotensin II (Ang II) and angiotensin III (Ang III), as well as
other peptide fragments in circulation are outlined in Fig. 4.1. The initial
event is the synthesis and release of the proteolytic enzyme renin that
cleaves angiotensinogen. In mammals, circulating levels of
angiotensinogen, constitutively secreted by the liver are reported to be one
thousand times or more greater than the normal concentrations of
circulating Ang II, the main physiologically active component of the
system (e.g., Klett and Granger, 2001; Lantelme et al., 2002), which has
been taken to suggest that hepatic angiotensinogen synthesis is not the
major rate-limiting step in determining circulating Ang II. Nevertheless,
plasma concentrations of angiotensinogen do appear to influence the
activity of the mammalian renin-angiotensin systems and physiological
results such as blood pressure elevation. This has been argued from a
biochemical point of view to reflect the fact that plasma concentrations of
angiotensinogen are within the concentration range of the Km value for
the enzyme substrate (renin-angiotensinogen) reaction (Klett and
Granger, 2001). Liver mRNA angiotensinogen and plasma
angiotensinogen have been reported to influence maximal angiotensin
formation (Brasier and Li, 1996; Klett and Granger, 2001), and Ang II
itself can regulate hepatic angiotensinogen mRNA, as well as renin mRNA
(Nakamura et al., 1990). Recent studies suggest significant regulation of
mRNA for angiotensinogen in the trout. After transfer of rainbow trout to
a hyperosmotic environment, hepatic angiotensinogen mRNA was
increased in parallel with rising plasma osmolality (Paley et al., 1996; Aust,
2002). These studies only became feasible after the cloning and
sequencing of angiotensinogen cDNA in the trout (Paley et al., 2003). The
87
Fig. 4.1 Renin-angiotensin and kallikrein-kinin systems showing simplified biochemical

cascades in circulation and the effects of enzymes, the ACE inhibitor, captopril, and the
vasodilator, papaverine. This diagram does not show the alternative pathways for possible
processing of angiotensinogen by angiotensinogenases and aminopeptidases that may
occur in tissues.
angiotensinogen gene (and protein) sequence has only been identified in

three teleostean fish species: the zebrafish, Danio rerio (GenBank Data
Base: AL772289; BC095585; NB198063), the pufferfish, Takifugu rubripes
(GenBank Data Base: BK001021) and the rainbow trout, Oncorhynchus
mykiss (GenBank Data Base: AJ579373). The rainbow trout and zebrafish
sequences show 64% identity, but have a very low homology with
mammalian angiotensinogens, except in the region encoding angiotensin
I, probably because of the redundancy of the majority of the molecule with
little need to conserve primary structure; the most important functional
parts of the molecule are the renin cleavage site and the angiotensin
I-containing region.
Renin is synthesized in differentiated smooth muscle cells of
vasculature supplying the renal glomerulus. This site of renin
production has long been considered the major source of circulating renin
for the systemic RAS. The classical mammalian picture of renin-producing
cells within the wall of the afferent glomerular arteriole has led to their
88
Fish Osmoregulation
description as juxtaglomerular cells, but in fish these cells often occur well
away from the glomerulus (Kobayashi and Takei, 1996). In marine fish not
possessing glomeruli, the aglomerular teleosts, juxtaglomerular cells would
clearly be a misnomer and granular epithelioid cells is a preferable and
more accurate term.
Identification of the cells producing renin in fish has often proved
more difficult than in mammals. Initial studies relied on complex
histological staining of the tiny renin granules. In mammals, these
approaches have now largely been overridden by immunohistochemistry
using specific renin antisera (e.g., Gomez et al., 1990). However,
mammalian antibodies rarely bind to fish renins and specific antisera are
still needed for research on fish. The sequencing of mammalian renin
genes has enabled the development of molecular probes to investigate
renin mRNA (Hackenthal et al., 1990), but amongst fish the renin gene
has as yet only been identified in the zebrafish, D. rerio and pufferfish,
Takifugu rubripes (Liang et al., 2004), hence impeding progress in
investigations of expression of the renin gene in most fish. Intriguingly,
abundant expression of renin mRNA in early development of zebra fish
has been demonstrated but the functional significance of this is as yet
unclear (Liang et al., 2004) and the specific cells expressing the renin gene
have not yet been identified.
Despite the lack of an ability to investigate renin expression in fish
other than the zebrafish and pufferfish, the kidney seems very likely to be
the major source of reninas in other vertebratesbut the existence of
additional sites of renin production, as part of tissue specific RAS, has been
known for many years in mammals (Hackenthal et al., 1990; Yanegawa et
al., 1991; Bader et al., 2001). In fish, there is less evidence as yet of tissuespecific RASs, but this should not be taken as indicating that they do not
exist; it is more likely to be merely a reflection of the lack of the appropriate
studies, rather than a lack of paracrine RASs. The first extra-renal tissue
that was revealed to synthesize renin in teleost fish was the corpuscles of
Stannius (CS), the small endocrine glands that lie on the surface of the
kidney or embedded within the kidney (Hasegawa et al., 1984). The CS
have been argued to contain a calcium-regulating hormone, although in
eel, renin secreted by the CS has been suggested to play a role in regulation
of cardiovascular function, rather than in calcium regulation (Butler and
Zhang, 2001; Butler et al., 2003). Renin, either in circulation or in tissuespecific systems, acts on the substrate, angiotensinogen, in order to form
89
the decapeptide, angiotensin I (Ang I), as shown in Fig. 4.1. It is, therefore,
important that recent studies have also identified angiotensinogen mRNA
in the CS (Aust, 2002), suggesting that they contain a complete RAS. The
role of this paracrine system is as yet unclear, but its involvement in
osmoregulation seems likely. Our recent studies of angiotensinogen gene
expression in trout also provide strong support for the occurrence of a
paracrine RAS in the kidney (Paley et al., 1996; Brown et al., 2000) and
brain (Aust, 2002). Once again, the roles of these systems are as yet
uncertain, but likely interaction with osmoregulatory processes will be
discussed in later sections of this chapter.
An important step in the systemic RAS cascade (Fig. 4.1) is the
conversion of Ang I to the octapeptide, Ang II, by angiotensin converting
enzyme (ACE), although in mammalian paracrine RASs such as that in
the heart, Ang II may be generated directly from angiotensinogen, through
the actions of cathepsin G, bypassing Ang I, and chymase may act as the
enzyme forming Ang II from Ang I (Nishimura, 2001). Angiotensin
converting enzyme, nevertheless, has a major role to play in converting
circulating Ang I to Ang II. ACE activity can be measured using the tripeptide substrate, hippuryl-histidyl-leucine, which is cleaved by ACE to
form His-Leu and hippuric acid. Most studies of ACE activity in fish
tissues have measured hippuric acid spectrophotometrically, but
fluorimetric assay of His-Leu offers both higher sensitivity and reliability
(Casarini et al., 1997; Cobb et al., 2002a, 2004). ACE-like activity has been
measured in all of the major groups of fish and for an extensive range of
tissue types, including kidney, heart, intestine, gill and brain (Lipke and
Olson, 1988; Uva et al., 1992; Cobb et al., 2002a, 2004). Much of the ACE
is membrane bound in vascular endothelial plasma membranes where the
long extracellular part of the molecule contains the active site, although
cellular mechanisms also regulate the secretion of soluble ACE into a
range of body fluids, including serum (Erdos, 1990; Ramchandran et al.,
1994; Casarini et al., 1997). Particularly high levels of ACE activity are
present in tissues with an extensive vascular endothelium, such as
mammalian, reptilian and amphibian lung (Lipke and Olson, 1988;
Bramucci et al., 2004), a feature likely to explain the high rate of
conversion of Ang I to Ang II as it passes through the pulmonary
circulation. In fish, the functional homologue of the mammalian lung, the
gills, has a similarly extensive vascular surface area and, in many species,
high ACE activity (see Cobb et al., 2004 for a review of the measurements).
90
Fish Osmoregulation
The respiratory lamellar pathway through the gills is a major site for Ang
I to Ang II conversion (Olson et al., 1989). However, other tissues, in
particular, the kidney and brain also have significant ACE activity
(Polanco et al., 1990; Uva et al., 1992; Cobb et al., 2002a).
The key role of ACE in the RAS has led to the development of
pharmaceutical inhibitors and allowed their use in physiological studies
aimed at investigating the actions of Ang II in fish. However, ACE
inhibitors such as captopril result in both a decline in Ang II and an
increase in circulating and tissue kinins, as ACE is identical to kininase II
which deactivates bradykinin (see Fig. 4.1), as well as cleaving other
peptides (Erdos, 1990). So, the action of kinins also needs careful
consideration. Nevertheless, ACE inhibitors have proved to be extremely
important tools in exploring the physiological role of the RAS of fish, as
we shall see in later sections of this chapter.
Not all of ACE is endothelial. For example, the brush border of the
renal proximal tubule epithelium contains high levels of ACE (Yanegawa
et al., 1991) that is likely to be linked to a paracrine RAS generating
nanomolar concentrations of intratubular Ang II (Navar and Nishiyama,
2004). Recent measurements of angiotensins in urine excreted from the
perfused kidney, where a hepatic supply of angiotensinogen does not exist,
suggests a similar renal RAS exists in teleosts (Aust, 2002).
ANGIOTENSIN SEQUENCES IN FISH
Over the past 25 years or so, angiotensins have been sequenced in all
major groups of fish, with the only exception now being the jawless hagfish
(Fig. 4.2). Until relatively recently, angiotensin had remained
unsequenced in lungfish and elusive in both cartilaginous elasmobranchs
and the jawless agnathan fish, but homologous incubations of kidneys and
plasma finally resolved the long-standing controversy as to the existence
of the renin-angiotensin system in elasmobranchs and lampreys
(Nishimura et al., 1970; Henderson et al., 1981; Takei et al., 1993, 2004;
Rankin et al., 2004). Angiotensin II in lampreys has proved to be identical
to that in most teleost fish, suggesting an early origin of this peptide
structure. Indeed, the structure of the physiologically active octapeptide,
Ang II is largely conserved throughout vertebrate groups, but with
interchange of the asparagine and aspartic acid at position 1 and valine
and isoleucine at position 5 in the agnathan lampreys, most teleosts and
the lungfish compared to mammalian species (Fig. 4.2). Recent searching

1
91
10
Arg
Val
Tyr
Ile
His
Pro
Phe
His
Leu
Arg
Val
Tyr
Val
His
Pro
Phe
Thr
Leu
Arg
Val
Tyr
Val
His
Pro
Phe
Thr
Leu
Asp
Arg
Val
Tyr
Val
His
Pro
Phe
Asn
Leu
Asn
Arg
Val
Tyr
Val
His
Pro
Phe
His
Leu
Asn
Arg
Val
Tyr
Val
His
Pro
Phe
Asn
Leu
Asn
Arg
Val
Tyr
Val
His
Pro
Phe
Asn
Leu
Asn
Arg
Val
Tyr
Ile
His
Pro
Phe
Thr
Leu
Asn
Arg
Val
Tyr
Val
His
Pro
Phe
Gly
Leu
Elasmobranchs
Dogfish
(Triakis scyllia)5
Asn
Arg
Pro
Tyr
Ile
His
Pro
Phe
Gln
Leu
Lungfish
(Neoceratodus
forsteri)9
Asn
Arg
Val
Tyr
Val
His
Pro
Phe
Thr
Leu
Mammals
human, horse, dog,
Asp
sheep, pig, rat, rabbit7
Agnathans
River lamprey
Asn
(Lampetra fluviatilis)11
Sea lamprey
Asn
(Petromyzon
marinus)12
Holosteans
Bowfin
(Amia calva)8
Teleosts
Japanese goosefish
(Lophius litulon)3
Chum salmon
(Oncorhynchus
keta)2*
Rainbow trout
(Oncorhynchus
mykiss)6
Flounder
(Platichthys flesus)10
Eel
(Anguilla japonica)1,
(A. rostrata) 4*
References: 1Hasegawa et al. (1983); 2Takemoto et al. (1983); 3Hasegawa et al. (1984); 4Khosla et
al. (1985); 5Takei et al. (1993); 6Conlon et al. (1996); 7see Kobayashi and Takei (1996); 8Takei et al.
(1998); 9Joss et al. (1999); 10Balment et al. (2003); 11Rankin et al. (2004); 12 Takei et al. (2004). *Asp 1,
Val 5-Ang also identified but unlikely to be naturally present; Asp1 and Asn1 interconvertible during
incubation.
Fig. 4.2 Angiotensin I and Angiotensin II amino acid sequences of peptides isolated from
fish species, compared to the peptide sequence in most mammals. Amino acids at
positions 3, 5 and 9 (Ang I only) that differ between species are highlighted. Further
differences occur in the dogfish and flounder.
92
Fish Osmoregulation
of angiotensinogen sequences in genome and EST databases has also

identified that Ang II in fugu, stickleback, medaka, carp and zebrafish are
identical to the typical (not flounder) teleostean sequence (Takei et al.,
2004). Intriguingly, elasmobranch angiotensin was found to be more
unusual, with asparagine at position 1 (as in teleost fish), isoleucine at
position 5 (as in mammals) and a unique proline substitution at position
3 (Fig. 4.2). Due to the large differences in the aliphatic side chains of
proline and valine, and the influence this has on protein architecture,
proline3 will profoundly affect the tertiary structure of Ang II (Creighton,
1993). This may well explain why some antibodies generated against
mammalian Ang II structure fail to recognize elasmobranch Ang II.
However, a homologous radioimmunoassay (RIA) for elasmobranch Ang
II has been established (Tierney et al., 1998) and basal circulating
concentrations in the European spotted dogfish, Scyliorhinus canicula
shown to be in the range of 100-150 pM, although these values may rise
up to ten fold during experimental manipulations (Anderson et al., 2002a).
Only in hagfish is an Ang II yet to be isolated. Lampreys and hagfish
are the sole survivors of a once flourishing radiation of jawless fishes.
Although they are still grouped together as cyclostomes, molecular
phylogenetic analyses suggest a diphyletic origin, with lampreys more
closely related to elasmobranchs and other jawed vertebrates, while
hagfish appear to be the survivors of a group that diverged from the main
craniate lineage at least 500 million years ago (Janvier, 1999). Granules of
renin could not be identified in the renal vasculature of the Japanese
hagfish (Paramyxine atami) or the Atlantic hagfish (Myxine glutinosa) and
incubation of renal extracts of P. atami with homologous or heterologous
plasma did not yield a pressor substance (Nishimura et al., 1970;
Nishimura, 1985), suggesting a lack of a RAS. However, recent
radioimmunoassay has detected immunoreactive angiotensins in hagfish
plasma, at around 250 pM (Cobb et al., 2004). The antisera employed has
a high binding affinity for Ang II and low cross reactivity with Ang I, but
high cross reactivity to Ang III and IV, so measurement of Ang II, III and
IV might be predicted. The concentration measured is in the same order
of magnitude as angiotensin concentrations reported in fish and
amphibians and the parallelism of serially diluted plasma with angiotensin
standards supports the existence of circulating angiotensin in hagfish,
although the sequence of this angiotensin clearly is yet to be determined.
93
ANGIOTENSIN RECEPTORS
Amongst teleost fish, angiotensin receptors have been identified in many
tissues (see Table 4.1) giving us clues as to probable sites of action,
including actions on osmoregulatory tissues. For example, binding of
angiotensin to the chloride cells of eel gills (Marsigliante et al., 1996,
1997a) led to research that has identified rapid actions on gill Na+,K+ATPase and a highly probable role in determining sodium balance. In
teleostean kidneys, Ang II binding sites have been identified in the renal
tubules of several species (Cobb and Brown, 1992; Marsigliante et al.,
1996, 1997b), suggesting actions on renal transport processes, but fewer
studies have identified glomerular receptors. No Ang II binding to the eel
glomerulus was detected by immunocytochemistry (Marsigliante et al.,
1996) although this may simply reflect failure of the monoclonal antibody
(selective to mammalian AT1 receptors) to recognize teleostean Ang
receptors. Radioligand-binding techniques have shown the occurrence of
an Ang II binding site in the renal glomerulus of the trout and
displacement studies suggest the receptor has some similarities to the
mammalian AT1 sub-type, although significant differences were revealed
in signalling studies (Cobb and Brown, 1993; Brown et al., 1997; Cobb
et al., 1999). The glomerular receptor of trout has been linked to renal
antidiuretic actions and this action will be discussed more fully later when
reviewing the renal actions of Ang II in fish.
The apparent cloning of a fragment of an Ang receptor gene from the
rainbow trout (Parkyn et al., 1997) was later revealed to encode a protein
with a higher homology to mammalian and Xenopus angiotensin receptorlike proteins (Aust, 2002), that do not bind Ang II, and despite further
cloning attempts the trout receptor remains elusive. However, cloning of
a cDNA encoding the entire angiotensin receptor in eel liver (Tran van
Chuoi et al., 1999) confirms a receptor with a higher homology to AT1
than AT2 receptors, although phylogenetic analysis separated the eel
angiotensin receptor from all mammalian AT1 receptors and AT2
receptors and the angiotensin receptors of the African clawed toad,
Xenopus (Sandberg, 1994).
In elasmobranch fish, autoradiographic and ligand-binding techniques
have indicated the presence of Ang II receptors in a range of tissues (see
Table 4.1) including brain, heart, intestine, gill, kidney rectal gland and
liver (Hazon et al., 1997a; Tierney et al., 1997a; Cerra et al., 2001),
although these studies were unable to determine receptor sub-types. More
Glomerulus
(O. mykiss)
Intestine; gill (chloride cells and pavement

cells); kidney tubules basolateral cytoplasm;
weaker on apical membranes
Icefish
(Chionodraco hamatus)
(Table 4.1 contd.)
Marsigliante et al. (1997b)
Qin et al. (1999)
Radioligand binding: Asn1, Val5-Ang II

Kd: 1 nM; Ca2+ signalling: fura 2
Vascular smooth muscle: ventral and dorsal

aortae, branchial arteries, coeliac artery
Toadfish
(Opsanus tau)
Immunohistochemistry
Tran van Chuoi et al.

(1998)
Marsigliante et al. (2000)
Ca2+ signalling: fura 2; inositol

phosphate generation
Cloning cDNA
Kidney tubules
(A. anguilla)
Marsigliante et al.
(1996, 1997a)
Marsigliante et al. (1994)
Immunocytochemistry and isoelectric

focussing gill membranes
Radioligand binding and isoelectric

focusing: Ang II Kd intestine3.4 nM
Brown et al. (1997)

Cobb et al. (1999)
Brown et al. (1990)

Cobb and Brown (1992)
Ca2+ signalling: calcium green

fluorescence
I-Asn1,
Cobb and Brown (1993)

Brown et al. (1997)
125
Reference
Radioligand-binding Asn1,
Val 5-Ang II: Kd = 0.32-0.39 nM;
Losartan (mammalian AT1
subtype inhibitor) IC 50: 27 nM
Autoradiography:
Val5-Ang II
Technique and receptor

affinity (Kd or IC50 )
(A. anguilla)
Gill: chloride cells, cytoplasm, membranes
(A. anguilla)
Intestine: brush border membrane liver

kidney tubules: brush border membrane
Glomerulus
(O. mykiss)
Eel
(A. anguilla)
Kidney: glomerulus and its vascular pole,

proximal tubule; urinary bladder; dorsal and
ventral aorta; intestine; oesophagus; liver;
skin; heart; interrenal tissue; gill; brain
Tissues with identified

receptors
Rainbow trout
(Oncorhynchus mykiss)
Teleosts
Fish group
species
Table 4.1 Angiotensin receptors or binding sites identified in fish tissues. The techniques employed in each study are listed here.
94
Fish Osmoregulation
Interrenal; gills; rectal gland; kidney; brain;

heart; intestine; liver
Heart
Rectal gland, interrenal gland
Kidney, rectal gland, interrenal tissue,

gill brain, liver, spleen, heart, intestine,
muscle
Japanese dogfish
(Triakis scyllia)
European spotted dogfish

(Scyliorhinus canicula)
(S. canicula)
(S. canicula)
Elasmobranchs
(Table 4.1 contd.)
Cerra et al. (2001)
Hazon et al. (1997a)
In vitro autoradiography 125I-Asn1, Pro3,

Ile5-Ang II binding sites in ventricle,
atrium and conus arteriosus; Ventricle:
2 sites Kd: 0.05 nM and 1.3 nM
In vitro autoradiography 125I-Asn1, Pro3,
Ile5-Ang II. Rectal gland sub-caspule
Ang II K d: 0.41 nM; Interrenal:
2 sites Kd: 0.25 nM and 7 nM
Cloning cDNA: homology to mammalian

Aust (2002)
AT 1A receptor (50%) and AT 2 receptors (38%).
mRNA angiotensin receptor expression
Tierney et al. (1997a)

Hazon et al. (1997a)
In vitro autoradiography and radioligandbinding 125I-Asn1, Pro3, Ile5-Ang II.

Gill cells: 2 sitesKd: 0.11 nM and 1.75 nM
95
96
Fish Osmoregulation
recently, cloning of a full-length cDNA for an angiotensin receptor in the

European lesser spotted dogfish, S. canicula, an elasmobranch fish, again
shows greater similarity to mammalian AT1 receptors (~50% identity)
than mammalian AT2 receptors (~38% identity) and phylogenetic
analysis groups the dogfish angiotensin receptor with AT1 receptors (Aust,
2002). Seventeen of the 20 amino acid residues identified as essential for
structural integrity, angiotensin binding and receptor internalization of
mammalian AT1 receptors are conserved in the dogfish receptor. However,
low conservation of extracellular loop 2, and non-conservative
substitution of the amino acid predicted to bind to the unique Pro3 of
dogfish angiotensin, provide evidence for a predictable co-evolution of the
peptide and its receptor.
Expression of angiotensin receptor mRNA in dogfish tissues and
ligand-binding studies in teleosts show the occurrence of angiotensin
receptors in brain, liver, spleen, heart, rectal gland (elasmobranchs only),
intestine, kidney, interrenal tissue, gill and muscle tissue, giving evidence
for a diverse range of physiological actions, including roles in
osmoregulation that will be examined later in this chapter.
EURYHALINITY AND ACTIVATION OF THE RENINANGIOTENSIN SYSTEM
There is evidence that the RAS of fish is activated in association with
exposure to higher salinities. Amongst the euryhaline teleosts, the species
most studied with regard to the RAS are the eel (European and Japanese
eel), flounder and rainbow trout. For example, circulating levels of Ang II
are three times higher in seawater (SW)-adapted European eel, Anguilla
anguilla (~33 fmol/ml) than in freshwater (FW) eels (Tierney et al.,
1995a). In the Japanese eel too, circulating angiotensin increased
transiently after transfer to SW (Okawara et al., 1987), but then returned
to basal levels (Okawara et al., 1987; Tsuchida and Takei, 1998), indicating
a lack of a persistent elevation and suggesting roles in short-term adaptive
function of osmoregulatory tissues. This pattern of response does not seem
to apply in the jawless lampreys.
Our understanding of the RAS of jawless cyclostomes and its potential
links to euryhalinity is mostly derived from work on the river lamprey,
Lampetra fluviatilis. Capture of lampreys in the Ringkbing Fjord, Denmark
at the start of their upstream migration, has allowed measurements of Ang
in lampreys acclimated for several weeks to a range of salinities, and after
97
transfer from one salinity to another (Rankin et al., 2001; Brown et al.,
2005). The first measurements of plasma Ang concentrations in river
lampreys separated Ang II and Ang III (see Fig. 4.1) by high-pressure
liquid chromatography (HPLC) (Rankin et al., 2001). Both Ang II and
Ang III were found to be significantly higher in SW-acclimated rather than
in FW-acclimated river lampreys. These first studies also suggested a
possible link between circulating Ang and rising plasma osmolality, and
recent investigations have shown changes in plasma Ang concentrations
after rapid changes in environmental salinity. Figure 4.3 shows a significant
decrease in plasma Ang after the decrease in environmental salinity,
alongside declining plasma osmolality and haematocrit, an index of plasma
volume in lampreys that lack a splenic source of stored red blood cells or
the secondary circulation that occurs in teleost fish (Brown et al., 2005).
Reduced RAS activity due to volume expansion in lampreys experiencing
an acute decrease in the external osmolality is in keeping with the rapid
rise in plasma Ang that was shown to occur after blood volume depletion
(Brown et al., 2005). Volume receptors are, therefore, likely to play a major
role in regulating circulating Ang in lampreys during their exposure to
different salinities.
In addition to volume receptors, osmo (sodium) receptors also appear
to play a role in regulating the RAS of river lampreys. The evidence for this
hypothesis comes from more than one experimental approach. Firstly, after
an acute increase in external salinity, rising plasma Ang occurred but
haematocrit was unaffected, indicating little change in plasma volume
(Fig. 4.3). Secondly, intraperitoneal injections of known volumes of saline
influence plasma concentrations of Ang in ways that suggest both volume
and osmo/salt receptors. Intraperitoneal injection of 1% by volume of body
mass of isosmotic saline led to a rapid reduction in circulating Ang that
was not apparent after injection of the same volume of hyperosmotic
saline, even though similar volume expansion was implied by the declining
blood haematocrit (Fig. 4.4). The increased plasma osmolality after
hyperosmotic injection suggests osmo/salt receptors interact with volume
receptors in regulating the lamprey RAS. In placing these signals in
context, with regard to the euryhalinity of lampreys, changes in plasma
volume and electrolytes when anadromous lampreys migrate between FW
and SW will act in a complementary fashion; high plasma volume and
lowering of plasma osmolality in FW would both act to depress the RAS
while reduced plasma volume and increased plasma osmolality would both
98
Fish Osmoregulation
Fig. 4.3 Effects of a rapid increase in environmental salinity (FW to 21 ppt; 605 mOsm
kg1) and a rapid decrease in environmental salinity (758 mOsm kg1 to 22 mOsm kg1) in
the river lamprey. Changes in (A) plasma osmolality (mOsm kg1), (B) blood haematocrit
(%), indicative of changes in plasma volume in lampreys, and (C) plasma angiotensin
concentrations (pM) are shown (n=7 to 9 at different time points). Values are means SEM.
Groups with different letters differ significantly (ANOVA and post hoc multiple comparison
tests). Asterisks indicate groups that differ significantly from time 0 h (* p<0.05, **p<0.01,
ANOVA and linear contrast analyses). Data presented in Brown et al. (2005).

A
Non-injected
45
Haematocrit (%)
40
Isosmotic
99
Hyperosmotic
a
35
30
b
b
25
b
20
15
15 min
30 min
Time after intraperitoneal injection

450
c
-1
Plasma Osmolality (mOsm kg )
400
350
300
a
ab
b
ab
250
200
15 min
30 min
Plasma Angiotensin (pM)
1000
800
a
600
a
a
a
ab
400
b
200
0
15 min
30 min
Fig. 4.4 Plasma angiotensin (pM) in river lampreys held in freshwater (Panel C), 15 min
and 30 min after intraperitoneal injection of 1% body mass by volume of isosmotic saline
(120 mM NaCl; 233 mOsm kg1; n=5 at each time point) or hyperosmotic saline (4M NaCl;
n=5 at 30 min; n=8 at 30 min). Control lampreys (non-injected) were held in light
anaesthesia for 15 min (n=8) and 30 min (n=4) in the absence of further manipulation.
Panel (A) shows haematocrit (%) and panel (B) shows plasma osmolality (mOsm kg1) of
these fish. Values are means SEM. Different letters above error bars signify groups that
differ significantly (ANOVA and post hoc multiple comparison tests). Data presented in
Brown et al. (2005).
100
Fish Osmoregulation
activate the RAS when meeting hyperosmotic environments. However,

lampreys feed on fish blood and tissues during the marine phase of their life
cycle, and this could also have major impacts that are not necessarily
complementary. Teleost blood would expand extracellular fluid volume
with an approximately isosmotic load, reducing plasma Ang, but sea
lampreys feed on sharks and this causes simultaneous volume and
hyperosmotic challenges (Wilkie et al., 2004). The implications of these
differences at a functional level are fascinating but as yet unexplored.
Although many elasmobranch fish only inhabit the marine
environment, there are true euryhaline elasmobranch fish that inhabit
both full FW and SW environments and many species exhibit some
capacity to acclimate to dilute SW, at least under laboratory conditions,
and may be regarded as partially euryhaline (Hazon et al., 2003). In SW,
fully euryhaline elasmobranch fish osmoregulate as stenohaline marine
fish by maintaining the body fluid osmolality slightly hyperosmotic to SW
primarily due to the hepatic and muscle (Kajimura et al., 2006) synthesis
(via the ornithineurea cycle) and renal retention of the nitrogenous
compound urea (Thorson et al., 1973; Piermarini and Evans, 1998). In FW,
body fluid osmolality is maintained hyperosmotic to the environment and
increased in comparison to teleosts (greater than twofold). This hyper/
hyper osmoregulatory strategy in FW/SW for euryhaline elasmobranch fish
is distinctly different to that employed by euryhaline teleosts. Until
recently, our knowledge of the osmoregulatory strategy of fully euryhaline
elasmobranchs was based largely on work carried out on the bullshark,
Carcharhinus leucas (Thorson, 1962, 1967; Thorson and Gerst, 1972;
Thorson et al., 1973). Studies have now been extended to include the
euryhaline Atlantic stingray, Dasyatis sabina (Piermarini and Evans, 1998;
Piermarini and Evans, 2000, 2001; Janech and Piermarini, 2002) together
with further studies of C. leucas (Pillans and Franklin, 2004; Pillans et al.,
2005). It is evident that there is a marked shift in the percentage
contribution that sodium, chloride and urea provide to plasma osmolality
in FW and SW environments. It appears that the euryhaline species, D.
sabina and C. leucas, although displaying reduced plasma osmolality in FW
compared to SW, maintain the capacity to retain urea as they acclimate to
reduced environmental salinities (Hazon et al., 2003). However, when
both fully and partially euryhaline elasmobranchs acclimate to
environments of higher salinity, plasma osmolality must increase rapidly so
that plasma osmolality is maintained iso- or slightly hyperosmotic to the
new environment. In this case plasma concentrations of sodium and
101
chloride increase before urea (Anderson et al., 2002b, 2006). The

implication of these findings will be discussed later in this chapter (see text
on p-109, Drinking).
There is now evidence that, as in teleost fish and lampreys, the RAS
in elasmobranchs is activated in association with exposure to higher
environmental salinities. In the partially euryhaline lesser spotted dogfish,
S. canicula, Ang II concentration increased after acute transfer from 80%
to 100% SW (Fig. 4.5; Anderson et al., 2002a). In the fully euryhaline
bullshark C. leucas, Ang II concentrations were also elevated during a
more long-term transfer from FW to SW that occurred over a period of
days (Anderson et al., 2005).
ROLES OF ANGIOTENSIN IN OSMOREGULATION
Angiotensin has a wide range of biological actions in different tissues.
Osmoregulatory actions generally appear to be fast acting and of relatively
short duration. However, the continual presence of significant circulating
levels of angiotensin in fish may result in longer-term regulatory effects.
Also, the stimulation of the release of longer-acting steroid hormones may
have prolonged effects on osmoregulatory processes. It is clear that in
Fig. 4.5 Plasma levels of Ang II (fmolml1) in S. canicula following rapid transfer from
80% SW to 100% SW (n=7). Statistical comparison was made between values taken at
time 0 h and immediately after transfer. Ang II was significantly increased within 90 minutes
of transfer. Values are means S.E.M; *p<0.05 (Students t-test). Taken from Anderson
et al. (2001a) (with permission).
102
Fish Osmoregulation
mammals both a systemic circulating RAS and local paracrine systems

exist and recent studies in fish are beginning to reveal similar complexities.
In the following sections, we will review the actions of Ang II in the
peripheral system and in paracrine systems and their regulation of
drinking, renal function, rectal gland (in elasmobranchs), gill and
interrenal function.
DRINKING
Amongst teleost fish, drinking is an important component of volume
homeostasis in a hyperosmotic environment where loss of water across the
gills is balanced by copious drinking of the environmental medium and
absorption of a large proportion of the water passing through the intestine.
Freshwater teleosts, already facing a continual osmotic water influx, were
initially thought to drink none of the surrounding freshwater, but a closer
examination showed this to be untrue (see Table 4.2: Kobayashi et al.,
1983; Fuentes and Eddy, 1997; Tsuchida and Takei, 1999), although
drinking of FW may normally be linked to food digestion rather than any
osmoregulatory requirement for water (Rankin, 2002).
The renin-angiotensin system was first revealed to play a prominent
role in the regulation of drinking in studies of rats in which ligature of the
vena cava (reducing cardiac output) stimulated drinking, but
nephrectomized rats showed no drinking response indicating that a renal
factor, believed to be renin, was necessary for the drinking response
(Fitzsimons, 1964). Further studies led to the idea that angiotensin is an
important physiological regulator of drinking (Fitzsimons and Simons,
1969; Fitzsimons, 1998). Angiotensin II induced drinking has now been
demonstrated in all vertebrate groups, with the only apparent exception so
far being the jawless fish.
Thirst and physiological drinking in mammals, birds and reptiles is
determined by three main stimuli: increasing plasma osmolality (and
linked cellular dehydration), decreased blood volume (= extracellular
dehydration), and an increase in Ang II (Fitzsimons, 1998). Drinking rates
in teleosts seem to be regulated primarily by extracellular dehydration
(blood volume) and Ang II, which in themselves can be linked (Nishimura
et al., 1979; Olson, 1992), with little evidence or no evidence of
stimulation of drinking by cellular dehydration after injections of
hypertonic saline (Beasley et al., 1986; Takei et al., 1988). A dipsogenic
action of Ang II was first demonstrated in two euyhaline teleosts, the eel
103
and the killifish (Hirano et al., 1978; Malvin et al., 1980). Since then,
dispogenic responses to Ang I (subsequently converted to Ang II) and/or
Ang II have been shown in many teleosts (Table 4.2).
An early study compared the effects of intraperitoneal injection of
Ang II on drinking in 37 species of teleosts, some considered stenohaline
FW species, some euryhaline species and a group of stenohaline marine
species, after adding phenol red to the environmental medium, as a marker
for drinking (Kobayashi et al., 1983). Many of the freshwater species did
not respond to Ang II, even though the circulating concentrations
reached are likely to have been at pharmacological levels, at least
transiently. Unresponsive species were Pseudorasbora parva (topmouth
gudgeon), Rhodeus ocellatus (rose bitterling), R. lanceolatus (slender
bitterling), Ctenopharyngodon idealla (grass carp), Cobitis anguillicaudatus
(Asian pond loach), Salvelinus leucomaenis (Japanese char), Pungitis sinensis
(Chinese stickleback), Oreochromis mossambicus (Mozambique tilapia) and
Cyprinus carpio (carp) and this list was later extended when a lack of a
drinking response to Ang II was reported in Natropis cornutus (common
shiner), Cottus bairdi (mottled sculpin) and Carassius auratus (goldfish)
(Beasley et al., 1986). The goldfish had showed a drinking response to Ang
II in Kobayashis studies (Table 4.2) which raises certain questions relating
to experimental protocols. The sequence of the Ang II employed is
particularly important. Asp1, Ile5-Ang II was used by Beasley et al. but is
unlikely to be the endogenous sequence (see Fig. 4.2). However, it has also
been argued that the strain of goldfish used by Kobayashi et al. (1996),
(that showed a drinking response to Ang II) was particularly saline
tolerant (Kobayashi and Takei, 1996). Since these studies, intramuscular
Ang I (which would be converted in vivo to Ang II) has also been shown
to induce a dispogenic response in carp held in freshwater (Perrott et al.,
1992; see Table 4.2), but the carp was a non-responder to Ang II in
Kobayashis studies, and this too may reflect differences in salinity
tolerance of the strains of carp employed. However, despite the drinking
response to Ang I in the carp held in freshwater, and the evidence that
inhibition of Ang I conversion to Ang II inhibited the drinking response,
an effect of angiotensin on drinking in carp could not be demonstrated
when carp were held in hyperosmotic brackish water (40% SW)
(Perrott et al., 1992). In these conditions, the carp increased their plasma
osmolality by almost 50%, but did remain hyposmotic to the environment.
This draws our attention to the importance of saline tolerance in any
104
Fish Osmoregulation
Table 4.2 Teleost and elasmobranch fish: Ang-II stimulated drinking in fish held in
freshwater (FW) or seawater (SW).
Species
Freshwater Teleosts
Carassius auratus 3
C. carassius3
Leuciscus hakonensis3
Gambusia affinis3
Gyrinocheilus anymonieri 3
Parasilurus asotus3
Tridentiger obscurus3
Cyprinus carpio8
Euryhaline Teleosts
Anguilla anguilla8
A. japonica3
A. japonica1, 5
A. japonica1
A. japonica11
A. japonica13
Oryzias latipes3
Platichthys flesus8
P. flesus4,6
Salmo salar10
Oncorhynchus mykiss9
Seawater Teleosts
Glossobius giuris
fasciatopunctatus3
Callionymus richardsoni3
Hypodytes rubripinnis 3
Chasmichthys gulosus3
Sillago japonica3
Mugil cephalus 3
Pseudopleuronectes
americanus7
Pleuronectes platessa8
Myxocephalus
octodecemspinosus 7
Myxocephalus scorpius8
Limanda limanda8
Merlangius merlangus 8
Environment
Ang II dose
(per g body wt)
Peptide sequence
ip 100 ng
ip 100 ng
ip 10 ng
ip 100 ng
ip 10 ng
ip 200 ng
ip 100 ng
im 0.3 nmol
Asn1,Val5-Ang
Asn1,Val5-Ang
Asn1,Val5-Ang
Asn1,Val5-Ang
Asn1,Val5-Ang
Asn1,Val5-Ang
Asn1,Val5-Ang
Val5-Ang I
im 0.3 nmol
ip 10 ng
min. ia 0.5 ng
min. ia 0.05 ng
vascular; 0.1-10 fmol/min
intracranial 0.25-25 pmol
ip 10 ng
im 0.3 nmol
iv 6, 150 ng/min
im 0.4 nmol
im 0.2 - 2 nmol
Val5-Ang II
Asn1,Val5-Ang II
Asp1 or Asn 1, Ile-Ang II
Asn 1,Ang II
Asn 1,Val5-Ang II
Asn 1,Val5-Ang II
Asn1,Val5-Ang II
Val5 - Ang II
Asn 1,Val5 -Ang II
Asn1,Val5-Ang II
Asn1,Val5-Ang II
SW
ip 50 ng
Asn1,Val5-Ang II
SW
SW
SW
SW
SW
SW
ip 1 ng
ip 5 ng
ip 20 ng
ip 100 ng
ip 500 ng
im 13 ng
Asn 1,Val5-Ang II
Asn 1,Val5-Ang II
Asn1,Val5-Ang II
Asn 1,Val5-Ang II
Asn 1,Val5-Ang II
Asp1,Ile5-Ang II
SW
SW
im 0.3 nmol
im 13 ng
Val 5-Ang II
Asp1,Ile5-Ang II
SW
SW
SW
im 0.3 nmol
im 0.3 nmol
im 0.3 nmol
Val 5-Ang II
Val 5-Ang II
Val 5-Ang II
FW
FW
FW
FW
FW
FW
FW
FW
FW & SW
FW
FW
SW
FW
SW
FW
FW
FW/SW
FW
FW
II
II
II
II
II
II
II
(Table 4.2 contd.)
105
(Table 4.2 contd.)
Seawater Elasmobranch
Scyliorhinus canicula12
Triakis scyllia12
SW
SW
ia 0.01 - 2 nmol/kg
ia 0.001 - 2 nmol/kg
Asn 1,Pro 3,Ile5-Ang II

Asn 1,Pro 3,Ile5-Ang II
ip = intraperitoneal; im = intramuscular; ia = intraarterial; FW = freshwater; SW = seawater

References: 1Hirano et al. (1978); 2 Malvin et al. (1980); 3Kobayashi et al. (1983); 4 Carrick and Balment (1983);
5
Hirano and Hasegawa (1984); 6 Balment and Carrick (1985); 7 Beasley et al. (1986); 8 Perrott et al. (1992);
9
Fuentes and Eddy (1996); 10Fuentes and Eddy (1997); 11Tsuchida and Takei (1999); 12Anderson et al. (2001a);
13
Kozaka et al. (2003).
separation of freshwater, marine and euryhaline species when comparing

Ang-II sensitive and Ang-II insensitive species, but the salinity tolerances
and physiological adaptability to saline waters of many, so-called
freshwater teleosts are not established. The fundamental separation
should lie in whether a teleost fish species can achieve hyposmoregulation
in saline waterswhich can only occur if a drinking response is attuned
to the volume loss resulting from the hyposmoregulationalong with
appropriate changes in intestinal, gill and kidney function to absorb the
gut fluid, reduce urinary loss of fluid and excrete excess salts. These
physiological processes occur in all marine teleosts. But, in a constant
marine environment, there will be a stable water loss. Kobayashi et al.
(1983) suggested that stenohaline marine teleosts do not show a drinking
response to Ang II after showing that several of the exclusively marine
teleosts: Sardinops melanosticata (Japanese sardine), Trachurus japonicus
(Japanese horse mackerel), Sebastes inermis (black rockfish) and Rudarius
ercodes (Japanese file fish), did not show dipsogenic responses to
intraperitoneal injections of Ang II. However, several species that are
usually considered stenohaline marine species: Myxocephalus
octodecemspinosus (long-horned sculpin), Pseudopleuronectes americanus
(winter flounder), Pleuronectes platessa (plaice), Limanda limanda (dab)
and Merlangius merlangus (whiting), have subsequently shown drinking
responses to Ang II injections (Beasley et al., 1986; Perrott et al., 1992; see
Table 4.2). Nevertheless, Ang II effects on drinking are likely to be most
important in responding to variable salinities and a need to continually
adjust drinking rates in euryhaline fish.
The drinking response to Ang II has been most thoroughly
investigated in the euryhaline flounder, Platichthys flesus, and in Japanese
and European eels (Hirano et al., 1978; Carrick and Balment, 1983;
Kobayashi et al., 1983; Balment and Carrick, 1985; Perrott et al., 1992;
Tierney et al., 1995a,b; Takei and Tsuchida, 2000; Kozaka et al., 2003),
106
Fish Osmoregulation
although many of these studies have been pharmacological in nature,

using high levels of angiotensin. To examine the effects of physiological
concentration of Ang II, intravenous infusions of low levels would be more
appropriate, but few studies of this nature have as yet been undertaken.
One such study infused 0.1 to 10 pmol/kg/min into volume replete, FWacclimated, Japanese eels (with low basal drinking rates) and showed that
even in FW eels, exogenous Ang II has a potent dipsogenic action in this
euryhaline species (Tsuchida and Takei, 1999).
An alternative, powerful approach that has been employed to
investigate the regulation of drinking in fish, rather than simply
administering angiotensin (or angiotensin receptor antagonists/agonists),
is the pharmacological manipulation of the endogenous RAS. The RAS of
teleosts is activated in response to blood volume depletion (Nishimura
et al., 1979; Olson, 1992), so one favoured approach has been the use of
papaverine, a smooth muscle relaxant (see Fig. 4.1). In eels, papaverine
treatment results in a prolonged lowering of blood pressure (Tierney et al.,
1995a). Papaverine treatment of both FW- and SW-acclimated eels was
found to increase circulating Ang II in the eel, presumably in response to
the observed hypotension, and stimulated drinking, but the drinking
response could be blocked by treatment with captopril, an inhibitor of Ang
I to Ang II conversion (Tierney et al., 1995 a,b). Similarly, administration
of sodium nitroprusside (a nitric oxide releasing agent that induces
hypotension in trout) stimulated drinking in Atlantic salmon fry held in
FW, and this drinking was abolished by the angiotensin converting enzyme
inhibitor, enalapril (Fuentes et al., 1996).
Inhibition of Ang I conversion to Ang II using ACE inhibitors alone
has had varied effects on drinking rates. Large doses of captopril (72 mg/kg
injected intramuscularly or intravenously) inhibited drinking in the
euryhaline SW-acclimated European eel (Perrott et al., 1992; Tierney et al.,
1995a) but in two marine species that do not enter FW, winter flounder
and longhorn sculpin, captopril had no effect on basal drinking rates, even
though Ang II stimulated drinking by these two species (Beasley et al.,
1986). Physiological manipulation of the RAS to regulate drinking rates in
stenohaline marine species, therefore, appears to have less prominent
effects than in euryhaline species.
In a study of the copious drinking of SW-acclimated Japanese eels,
slow infusion of captopril at 0.01 or 1 mg/kg/min significantly reduced
drinking rates, even though Ang II was only significantly reduced at the
107
highest dose (Takei and Tsuchida, 2000). This could mean that captopril
at low doses achieves its effects through action on a brain RAS, and further
work is needed to clarify whether a brain RAS exists in the eel or not.
However, effects of captopril administration may be confounded by the
actions of bradykinin that we might expect to increase during ACE
inhibition, due to inhibition of bradykinin breakdown too (see Fig. 4.1).
Bradykinin has been shown to inhibit drinking in SW-acclimated eels,
independent of any change in blood pressure (Takei et al., 2001), so
depressed drinking rates after capropril treatment could simply reflect the
actions of elevated bradykinin.
Although experiments with captopril do not enable a clear distinction
of the effects of any brain RAS from those of peripheral angiotensin, an
alternative experimental strategy, the infusion of an antiserum to Ang II
into seawater-adapted eels, neutralizing the effects of free circulating
Ang II, has added to the picture (Takei and Tsuchida, 2000). While plasma
Ang II was dramatically reduced by this procedure, declining from more
than 60 fmol/ml to undetectable levels, drinking rates did not decrease
significantly. This suggested that the normal copious drinking of the SWacclimated eel does not rely simply on circulating angiotensin, even
though intravascular administration of angiotensin can exert a potent
dipsogenic action in the eel (Tsuchida and Takei, 1999).
The dipsogenic action of Ang II in teleosts raises questions as to the
localisation of Ang II receptors that result in drinking. In mammals, three
circumventricular organs (CVOs), the subfornical organ (SFO), the organ
vasculosum of the lamina terminalis (OVLT) and the area postrema (AP),
highly vascularized brain centres that lie outside the blood brain barrier,
have each been implicated in responding to circulating Ang II (Fitzsimons,
1998). For example, in rat the SFO responds to femtomole amounts of Ang
II injected directly into the tissue, while destruction of this tissue blocks
drinking responses to intravenous Ang II. However, in teleosts brain
lesions appeared to have relatively little effect on Ang II-induced drinking
responses (Hirano et al., 1978) and led to the suggestion that Ang II in
teleosts acts at the level of the medulla oblongata to induce swallowing
(Takei et al., 1985). In keeping with this idea, specific Ang II binding sites
have been identified in the medulla oblongata of the rainbow trout,
although further binding sites were also identified in the cerebellum (Cobb
and Brown, 1992). In this autoradiographic study, estimates of receptor
density were similar for trout acclimated to FW and SW, but as yet there
108
Fish Osmoregulation
has been no characterization of these receptors, which may differ in

binding affinities. In fish species that are restricted to FW over their entire
life span, it is feasible that on an evolutionary timescale receptors
responsible for inducing the drinking response have been lost, and that
this accounts for the apparent lack of a dipsogenic response to Ang II in
many FW species.
In mammals, distinct Ang II receptors have also been identified within
the blood brain barrier, and argued to be inaccessible or less accessible to
circulating Ang II, instead forming a component of an exclusively brain
RAS. The brain RAS is also thought to participate in controlling water
intake, as well as other physiological actions of Ang II such as blood
pressure regulation (Bader et al., 2001). Angiotensin II immunoreactivity
and binding sites are widely distributed in the mammalian central nervous
system and their actions may include brain remodelling and cell
differentiation that could include long-term changes in neuronal
organization affecting such aspects as sodium appetite (Fitzsimons, 1998).
Specific receptors for Angiotensin in the brain have been indicated by
recent studies of SW-acclimated eels in which angiotensin was injected via
a cerebral cannula inserted into the fourth ventricle. Intracranial Ang II
rapidly stimulated drinking (Kozaka et al., 2003). Intravenously injected
Ang II that might only have access to brain areas lacking a blood brain
barrier (believed to include the magnocellular preoptic nucleus and
anterior tuberal nucleus of the hypothalamus and the area postrema of the
medulla oblongata) also enhanced water intake in eels (Tsuchida and
Takei, 1999; Ando et al., 2000; Kozaka et al., 2003). However, comparison
of the time course of the effects of intracranial and intravascular Ang II on
drinking showed that intracranial Ang II exerts a more persistent effect
than circulating Ang II (Kozaka et al., 2003). The authors argued that this
might reflect the release of atrial natriuretic peptide in response to
circulating angiotensin that inhibits drinking in the Japanese eel (Takei,
2000). Thus, endogenous stimulation of drinking in euryhaline teleosts is
perhaps more likely to involve local release of Ang II from a brain RAS.
Although there is no direct evidence of a brain RAS as yet in the eel, the
evidence of a brain RAS in the rainbow trout does support this idea.
Until fairly recently, it was thought that elasmobranch fish did not
drink (Kobayashi et al., 1983). However, this assumption was based largely
on elasmobranchs acclimated only to full SW where plasma osmolality is
maintained iso- or slightly hyper-osmotic to the surrounding SW and
109
a small but constant influx of water means there is no physiological

requirement to drink. Furthermore, these early studies employed phenol
red as the marker for drinking rate and this is now regarded as a relatively
insensitive technique. For example, Kobayashi et al. (1983) reported a lack
of drinking response in the Japanese dogfish, Triakis scyllia. However, using
the sensitive oesophageal cannulation technique Anderson et al. (2001a)
demonstrated that T. scyllia does indeed drink and drinking rate increases
in response to transfer to higher salinity environments (Anderson et al.,
2002c). On entering environments of higher salinity, plasma osmolality of
both fully and partially euryhaline elasmobranchs must increase rapidly as
plasma osmolality is maintained iso- or slightly hyper-osmotic to the new
environment. The first report of drinking in elasmobranch fish during
acclimation to increased environmental salinity was in the European
spotted dogfish, S. canicula (Hazon et al., 1997b). Subsequently, Anderson
et al. (2002c) demonstrated that this drinking response was maximal
approximately 2 hours after transfer from 80 to 100% seawater and
resulted from extracellular dehydration (Anderson et al., 2002b), as in
teleost fish (Takei et al., 1988) and not cellular dehydration, as occurs in
mammals (Fitzimons, 1998). Indeed, cellular dehydration induced by
injection of hyper-osmotic sodium chloride-rich Ringer caused an
inhibition in the drinking response in S. canicula (Anderson et al., 2002b).
As discussed earlier, the rapid increase in plasma concentrations of sodium
and chloride before plasma urea concentrations increase suggests that
imbibed fluid is rapidly reabsorbed in the gastointestinal tract in order to
raise plasma osmolality. This response maintains the iso-hyper
osmoregulatory strategy as part of the acclimation response to entering
environments of higher salinity. However, to date, there is no direct
information of gut absorption of imbibed fluid in elasmobranchs. It is
evident, therefore, that elasmobranchs do have the physiological capacity
to drink and will do so when there is a requirement to rapidly increase
plasma osmolality following transfer to a hyper-osmotic medium.
The first evidence that the RAS was involved in the control of the
dipsogenic response of elasmobranchs was the demonstration that
administration of papaverine, a smooth muscle relaxant that stimulates
the endogenous RAS (Fig. 4.1), caused a significant increase in the
drinking rate in S. canicula (Hazon et al., 1989). Furthermore, the ACE
inhibitor captopril, when co-administered with papaverine, reduced the
papaverine-induced drinking response to basal levels. However,
110
Fish Osmoregulation
heterologous Ang II administration did not alter basal drinking rates.

Recently, homologous Ang II has been shown to induce a dose-dependent
increase in drinking rates in both S. canicula and in the Japanese dogfish
T. scyllia (Table 4.2; Anderson et al., 2001a). The drinking response was
further investigated in these two species of fish after acclimation to 80%
SW and then rapid transfer to 100% SW (Anderson et al., 2002a). Plasma
sodium and chloride concentrations increased rapidly within 6 hours
although plasma urea concentrations remained unaltered. The drinking
rate, determined by oesophageal cannulation (Anderson et al., 2001a), was
significantly increased, reaching maximal rates within 2-3 hours posttransfer. Plasma concentrations of Ang II increased rapidly, reaching a
maximum concentration 90 minutes after transfer to 100% SW
(Fig. 4.6). The time course of the Ang II response was similar to, but just
preceded, the increase in drinking rate (Anderson et al., 2002c). It is clear,
Fig. 4.6 Short term assessment of drinking rate in S. canicula following administration of
2 nmol/kg homologous Ang I ( ), Ang II ( ) and papaverine 10 mg/kg ( ). In the first 20 min
post-injection drinking rates in all 3 treatments were significantly higher than basal rates
***p<0.001. Drinking rates after Ang I and Ang II treatment remained significantly higher
than basal for 60 min post-injection, ***p<0.001 (n>6). Between 20 and 40 min, drinking
rates after papaverine treatment were significantly lower than basal levels **p<0.01 (n = 9,
7 and 8 for the respective treatments). Values are means + SEM. From Anderson et al.
(2001a) (with permission).
111
therefore, that the RAS is closely involved in the drinking response of

elasmobranch fish.
Drinking has also been demonstrated in lampreysboth the sea
lamprey, Petromyzon marinus and the river lamprey, Lampetra fluviatilis
(Rankin, 2002). L. fluviatilis acclimated to ocean strength seawater (1015
mOsm kg1) drank at 7.4 0.9 ml kg1 h1 (n=6) while FW-acclimated
river lampreys, that experience osmotic water influx, only drank 0.08
0.04 ml kg1 h1. Drinking would, therefore, appear to be an essential
component in the volume homeostasis of lampreys. Drinking in SWacclimated lampreys was coupled to absorption of more that 99% of the
sodium in the intestine (Rankin, 1997) and is likely to drive water
absorption by processes similar to those now well known in teleosts.
However, unlike those in teleosts, studies so far have failed to demonstrate
any effects of the RAS on drinking in lampreys. Kobayashi et al. (1983)
initially found no increase in drinking by FW-acclimated lampreys after
Ang II injections. This could reflect an inability to reinstate marine
osmoregulatory mechanisms after migration to FW where progressive gut
degeneration occurs, but further studies involving lampreys caught early in
their upstream migration and transferred back to hyperosmotic brackish
water so as to reinitiate drinking, still failed to reveal any evidence of
angiotensin effects on drinking (Rankin et al., 2001). The circulating
concentrations of angiotensin achieved in these studies were not
measured, but the results do suggest limited or no effect of angiotensin on
drinking. Papaverine, the smooth muscle relaxant that has been used so
effectively to demonstrate drinking linked to volume regulation in
elasmobranchs and teleosts (Hazon et al., 1989; Perrott et al., 1992;
Tierney et al., 1995b) did not stimulate drinking in lampreys acclimated to
50% SW, and in fact appeared to inhibit drinking (Rankin et al., 2001).
The effects on blood pressure were not explored; so papaverine may not
have worked effectively in this experiment. However, injection of the ACE
inhibitor, captopril, that inhibits conversion of Ang I to Ang II, did reduce
drinking rates of lampreys acclimated to 50% SW (Rankin et al., 2001).
Although the action of captopril on bradykinin formation and its
inhibition of drinking in teleosts (Takei et al., 2001) must be borne in mind,
further studies are warranted. As things stand, the evidence for
angiotensin action on drinking in lampreys is weak, but further work is
warranted.

Fish Osmoregulation
Drinking, as a component of the osmoregulatory mechanisms to

achieve volume homeostasis, is clearly of the greatest importance in fish
that suffer osmotic water loss, and for this reason, might be considered of
minimal importance in hagfish that are virtually isosomotic to a marine
environment. Early studies suggested that hagfish do drink their
environmental medium, but found no evidence for water absorption from
the gut (Morris, 1960), suggesting that drinking, plays a minimal role in
fluid balance in hagfish. However, even in this environment, fluid loss in
the small amounts of urine that are excreted does need replacement, and
drinking (as seen in teleosts and elasmobranchs) would be one way to
achieve this. Further research has confirmed that hagfish (inshore hagfish,
Eptatretus burgeri) drink their environmental medium, but found no
evidence of any action of angiotensin on drinking rates (Kobayashi et al.,
1983).
To be effective in acquiring water, drinking responses need to be
accompanied by functional changes in the gut to achieve solute-linked
water absorption. Recent study indicates that bicarbonate secretion in the
marine teleost intestine also plays an important role in facilitating water
absorption (Wilson et al., 2002). Angiotensin II ligand-binding sites appear
to be present in the teleost intestine (Cobb and Brown, 1992), so Ang II
may play a role in regulation of transport processes to maximize water
absorption. This idea has yet to be examined in fish, but is in keeping with
the evidence that Ang II increases sodium and water transport across all
regions of the mammalian intestine (Jin et al., 1998) and stimulates an
alkaline secretion in the duodenum (Johansson et al., 2001). If similar
actions exist in teleost fish, they could play an important part in SW
acclimation.
RENAL FUNCTION
The primary role of the kidneys in freshwater teleosts is to excrete the
excess water resulting from osmotic water influx while retaining solutes.
Glomerular filtration rates (GFRs) in FW teleosts are, therefore, typically
high, and only a low proportion of the filtered water (usually less than half,
but sometimes as little as a few %) is absorbed in the renal tubules (Rankin
et al., 1985; Brown et al., 1993). In contrast, GFR in SW teleosts is typically
much lower than that of FW teleosts and filtration is followed by an
increased reabsorption of water with tubular secretion of calcium,
magnesium, sulphate and phosphate (Brown et al., 1993; Pelis and Renfro,
2004) to produce a more concentrated urine, usually approaching the
overall osmotic concentration of plasma and rich in divalent ions.
!
The first evidence that angiotensin plays a role in regulation of renal

functional changes in euryhaline teleosts was obtained from experiments
involving intravenous infusion of Ang II into anaesthetized rainbow trout
(Brown et al., 1980). The profound antidiuretic action of Ang II in these
experiments was found to be primarily due to a reduction in GFR. Specific
binding sites for Ang II in the trout glomerulus and associated with the
vascular pole were subsequently demonstrated (Brown et al., 1990; Cobb
and Brown, 1993).
The reduced GFR in trout receiving exogenous Ang II could in theory
reflect reduced individual (single nephron) filtration rates (SNGFRs) or a
reduction in the number of actively filtering glomeruli, although the latter
are the most usual cause of changes in GFR in teleosts (Brown et al., 1978,
1993; Rankin et al., 1985). Changes in glomerular ultrastructure occur
after Asn1, Val5-Ang II infusion of freshwater trout, with a reduced
interdigitation of epithelial pedicels and flattening of the epithelial
podocytes (Gray and Brown, 1987) and the resultant ultrastructure is
reminiscent of the appearance of glomeruli in SW-acclimated trout
(Brown et al., 1983). However, SNGFRs appear to be higher in SWacclimated trout than FW-acclimated trout, and angiotensin infusion had
no effect on the SNGFRs of FW-acclimated trout, even though overall
GFR was reduced by 50% (Brown et al., 1980), so structural changes do
not seem to result in a reduced ultrafiltration coefficient, thereby
decreasing SNGFR. Instead, Ang II treatment increases the proportion of
non-filtering glomeruli (Brown et al., 1980, 1993). Direct evidence of this
action was obtained through the use of sodium ferrocyanide (a marker for
GFR that can be visualized after formation of Prussian blue, revealing the
filtering nephrons) by a method originally devised by Hanssen in 1958, but
that gained wide use in renal studies after Baines and de Rouffignac
developed the technique in the early 1960s so as to obtain measurements
for individual (single nephron) filtration rates. However, the original, nonquantitative Hanssen approach has been the most widely used method
(Aukland, 2001). In the rainbow trout, this technique provided direct
evidence that Ang II reduces the filtering population of nephrons and
comparison of the proportion of filtering nephrons in FW- and SWacclimated fish suggested that Ang II is important in regulation of
glomerular intermittency and renal responses to water availability.
Experiments using ferrocyanide also suggested reduced blood flow to
individual glomeruli (assessed qualitatively by the intensity of Prussian
"
Fish Osmoregulation
blue precipitation after ferrocyanide infusion), presumably, in the extreme,

leading to the cessation of filtration in many of the nephrons (Brown et al.,
1980, 1993).
In considering the studies that have employed infusions of exogenous
peptides to examine the renal action of Ang II in teleosts, it is essential to
appreciate the complications that may arise. Angiotensin is a powerful
vasoconstrictor in all vertebrates (Kobayashi and Takei, 1996) and this
action may confuse interpretation of the renal effects of Ang II,
particularly as in fish, renal function is not autoregulated, in contrast to
the position in mammals (Brown et al., 1993). Therefore, in teleosts,
changes in blood pressure often (but not always) result in parallel changes
in GFR and urine output (Nishimura and Sawyer, 1976; Brown et al.,
1993). As a result, prolonged infusion of Ang II in trout resulted in an
initial antidiuresis that was then abated by the coincident pressor action
(Gray and Brown, 1985). This may explain why some of the early studies
of the renal effects of Asn1, Val5-Ang II in both the lungfish, Neoceratodus
forsteri, and the American eel, Anguilla rostrata suggested a diuretic action;
an intraarterial infusion of exogenous Ang II was reported to result in a
diuresis in the American eel as a result of increased GFR but effects only
occurred after clear vasopressor responses to 10 and 100 ng/kg/min
(Nishimura and Sawyer, 1976).
An additional complication in interpreting results obtained so far in
whole animal studies is that circulating concentrations of Ang II during
intravenous infusions of Ang II or after injections of Ang II have not been
determined, so it is not possible to be certain that physiological effects
have been examined. However, use of an alternative in vitro approach, the
in situ perfused kidney that was developed in the early 1990s by Dunne and
Rankin, has added to the picture, and enabled the effects of physiological
concentrations of Ang II to be investigated. Using this preparation,
physiological concentrations of Ang II (Dunne and Rankin, 1992) and the
heptapaptide, (angiotensin 2-8) Ang III (Brown and Balment, 1997; Pope,
2002) have been shown to be antidiuretic. Ang III at 1011 M decreased
urine production by 22% while 109 M decreased urine output by 74 %.
These studies support the hypothesis that Ang II exerts an antidiuretic
action on the teleost kidney and may play an important role in the
antidiuresis that occurs in acclimation to hyperosmotic environments. In
addition to an antidiuretic action of circulating Ang II, studies with the
perfused kidney indicated stimulation of magnesium secretion (Dunne
and Rankin, 1992).
#
Tubular actions of Ang II were also suggested by studies in aglomerular

teleosts. In the aglomerular goosefish, Lophius americanus, Ang II was
reported to be diuretic and natriuretic (Churchill et al., 1979) and these
effects cannot, in aglomerular fish, reflect a direct link between blood
pressure and glomerular filtration! It was suggested that this action is likely
to reflect inhibition by Ang II of the reabsorption of sodium chloride and
the osmotic water reabsorption. This is contrary to the significant increase
in water reabsorption that accompanies the glomerular antidiuretic action
of Ang II in the glomerular trout kidney (Brown et al., 1980). In further
studies conducted on the aglomerular goosefish, inhibition of ACE to
lower circulating Ang II resulted in a decrease in urine output and
increased excretion of potassium, magnesium and calcium without
affecting blood pressure (Churchill et al., 1985). This supports the concept
that endogenous Ang II is diuretic in aglomerular fish independent of
actions on blood pressure, although changes in intrarenal blood flow and
its distribution cannot be ruled out. Actions of Ang II on tubular transport
processes are a possible explanation, but direct studies of the actions of
Ang II on renal tubular transport processes in either glomerular or
aglomerular teleost nephron have yet to be performed.
In the past 5 years, increasing evidence for a complete renal RAS that
has the potential to operate independently of the systemic system has been
obtained in trout. Parts of a renal RAS in fish had long been identified
within the kidney, as this organ is the established prime site for renin
synthesis. Added to this, many studies have reported angiotensin
converting enzyme activity in teleost kidneys (reviewed in Cobb et al.,
2004) and our recent research has demonstrated expression of the ACE
gene in the trout kidney (Paley et al., 1998). Demonstration of
angiotensinogen mRNA in kidney tissue provided the final element for a
complete renal RAS (Brown et al., 1995, 2000; Aust, 2002). More recently,
extraction of angiotensin from kidney homogenate after in vitro perfusion
of the trout trunk has added weight to the hypothesis that an active
intrarenal RAS occurs in trout (Brown, Aust and Frankling, unpublished).
The first studies aimed at investigating the actions of the renal RAS
of trout, used the in situ perfused trout kidney preparation treated with
captopril (Brown et al., 2000). This preparation excludes the liver and so
precludes existence of hepatic angiotensinogen and the systemic RAS.
Captopril treatment (to inhibit Ang II formation) in preparations perfused
$
Fish Osmoregulation
at a normal pressure head (38 cm H2O) had no effect on renal function,

but when preparations were perfused at a sub-physiological pressure of
25 cm H2O, which reduced perfusate delivery rates, captopril treatment
resulted in a glomerular diuresis. These results suggest firstly that the
intrarenal RAS in teleost fish is activated by low vascular pressure and/or
blood flow rates, and secondly that the angiotensin formed by the
intrarenal RAS plays an important role in regulating urine production.
The increased GFR during captopril treatment implies possible vascular
actions of a renal RAS that would in vivo presumably work hand in hand
with the established vascular actions of circulating Ang II. However,
further work is essential before the role and action of the renal RAS in fish
and how it operates alongside the systemic RAS can be fully appreciated.
Further work gives several lines of evidence that suggest renal tubular
actions of an intrarenal RAS in teleosts and it may be here that the
distinction from the actions of circulating Ang II appear. Firstly, in situ
hybridization has localized angiotensinogen mRNA in the proximal
tubules of the rainbow trout, suggesting that this is an important site for
Ang II formation. Secondly, measurable amounts of angiotensin have been
collected in urine produced by in situ perfused trout trunks (Aust, 2002).
Since proteases within the renal tubule and collecting ducts will degrade
the majority of intratubular angiotensin, measurable urinary
concentrations suggest that high concentrations are likely to occur in
tubular fluid in vivo, and act on the apical membrane of the tubular
epithelium. In the mammalian kidney, nanomolar concentrations of
Ang II occur in tubular fluid (Navar and Nishiyama, 2004) compared to
pM levels in circulation. Current evidence from the trout leads us to
believe that there was an early evolution of this control system, with
teleosts possessing an intratubular RAS. Binding of 125I-Ang II to trout
tissues suggests Ang II binding sites in the renal tubules (Cobb and Brown,
1992) and, in the eel, a renal tubular apical binding site specific for Ang
II was reported (Marsigliante et al., 1994). At the time of these studies, it
was envisaged that these receptors would bind circulating Ang-II after
filtration, but in the light of the recent demonstration of an intrarenal RAS
it seems more likely that Ang II generated within the tubular fluid will act
on the apical membrane ligand binding sites.
In early studies, intravenous Ang II infusion increased the renal
tubular water reabsorption of FW-acclimated trout, increasing the urine to
plasma concentration ratio of iothalamate, the GFR marker employed
%
(Brown et al., 1980). Whether this action reflected the effects on the basal
or apical epithelium could not be determined from the whole animal
studies. To obtain a clear picture of renal tubular actions of Ang II in
teleosts we now require more detailed studies such as microperfusion of
teleost nephrons to specifically examine the actions of Ang II and Ang III
on epithelial transport processes that may result in vivo from circulating
Ang II, or Ang II generated within the kidney, including within the renal
tubules. Of the few studies so far, one has examined the effects of Ang II
on Na+, K+-ATPase activity after administration of Ang II to perfused
kidneys and after exposure of isolated renal cells to Ang II (Marsigliante
et al., 2000). Both approaches showed that Ang II stimulates Na+, K+ATPase in FW-acclimated eels, in a dose-dependent way, with maximal
1.8 increase in the presence of 100 nM Ang II, but with no effect in SWacclimated eels, where Na+, K+-ATPase activity was already elevated.
Renal tubular Na+, K+-ATPase is involved in sodium transport across the
basolateral border of the renal tubules and in SW-acclimated fish leads to
water reabsorption (with sodium being regulated by the gills). Hence,
the effects on Na+, K+-ATPase activity reported in eels may explain the
earlier observations of increased renal tubular water absorption in
the trout.
Marine elasmobranch fish produce urine that is hypoosmotic to blood
plasma and the kidney is not regarded as a site for net excretion of sodium
and chloride: this is the role of the rectal gland. The elasmobranch kidney
consists of a series of nephrons that are both structurally and functionally
highly complex (Lacy and Reale, 1991a, b, 1995; Hentschel et al., 1993;
Lacy et al., 1995). Marine elasmobranchs maintain their plasma osmolality
slightly higher than that of the surrounding environment primarily due to
the hepatic and muscle synthesis and renal retention of the nitrogenous
compound urea. As more than 90% of filtered urea is reabsorbed, the
major role of the elasmobranch nephron appears to be urea retentive,
although much further work is required to elucidate the full functions of
the nephron. One of the major reasons for this lack of knowledge is that
renal studies in marine elasmobranch fish are technically difficult to
perform, at least in part, due to the very long and convoluted urinary
sinuses. Establishing basal urine flow ratesa prerequisite for in vivo renal
studiesis almost impossible without extremely long periods of urine
collection. In order to avoid some of these complications and to determine
the actions of individual peptides on kidney function, a perfused trunk
&
Fish Osmoregulation
preparation with the kidney in situ was developed for elasmobranch fish
(Wells et al., 2002) based on a technique previously described in teleosts
(Dunne and Rankin, 1992; Amer and Brown, 1995). To investigate the
possibility that the RAS in elasmobranchs may be involved in control of
GFR, the in situ trunk preparation in S. canicula was perfused with 109 M
Ang II and resulted in a significant decrease in urine flow rate and GFR.
This was associated with a decrease in the filtering population of nephrons
as determined by the ferrocyanide technique (Wells et al., 2005). The renal
effects of Ang II did indeed appear to be glomerular as no significant
changes were observed in any of the tubular parameters measured (Wells
et al., 2005). These results suggest that RAS is important in the control of
GFR as previously proposed for teleosts (Brown et al., 1980).
Ang II has been shown to have profound vasoconstrictor effects in
elasmobranchs (Tierney et al., 1997b; Hamano et al., 1998) and this may,
at least in part, account for the effects in the kidney. Glomerular bypass
shunts have been identified in S. canicula (Brown and Green, 1992) and
it is possible that Ang II may act at these shunts to vary the degree of
glomerular perfusion and control the proportion of active glomeruli.
Tierney et al. (1997a) reported the presence of Ang II receptors in the
elasmobranch kidney as has been previously reported for teleost fishes
(Cobb and Brown, 1993), although the precise location of these receptors
remains to be established. Preliminary evidence has also been presented
for an intrarenal RAS in the dogfish, S. canicula (Wells et al., 2003) that
is likely to play a role in regulation of renal function.
As in teleosts and elasmobranchs, Ang II appears to exert an
antidiuretic action in the lampreys, although the effects appear less
pronounced than in the teleosts (trout) or elasmobranchs (dogfish).
Indeed, initial studies reported a lack of any renal effect of Ang II injected
into the caudal vein of river lampreys adapted to a brackish medium
(Rankin et al., 1985), but this may have been because urine flow rates were
already very low, and further reduction hard to discern. Later studies in
freshwater-acclimated lampreys indicated an antidiuretic action (Rankin,
1997). More recent research has suggested that the pressor action of
exogenous Ang II (Rankin et al., 2004) may compromise the detection of
an antidiuretic action since lampreys, like teleosts, cannot autoregulate
their GFR. Increased blood pressure results in an increased GFR, but in
lampreys the glomerular diuresis results from an elevation in individual
nephron filtration rates, rather than an increase in the filtering population
'
of glomeruli, as there is a lack of glomerular intermittency (Brown and

Rankin, 1999). In both sea lamprey, Petromyzon marinus and the river
lamprey, Lampetra fluviatilis, acclimated to FW after capture during
upstream migration, only high doses of Ang II (1010 to 4 109 moles
min1 kg body wt1 infused intravenously) were antidiuretic (Cobb et al.,
2002b), and no changes in urine composition were identified. The
circulating concentrations achieved in these experiments would be likely
to have exceeded physiological levels, so the significance of Ang II in
regulating renal function in river lampreys remains unclear. An
antidiuretic action would be most appropriate in marine lampreys where
renal water conservation plays an important role in osmoregulation
(Rankin, 1997), but experiments that manipulate the RAS and explore
renal function in lampreys acclimated to SW have yet to be performed.
RECTAL GLAND
Elasmobranch fish possess a unique gland, the rectal gland that secretes a
fluid isosmotic to blood, but essentially composed of sodium and chloride
(Shuttleworth, 1988). The rectal gland is highly vascularized and
secretion rates of the gland are highly dependent on blood flow to the
secretory epithelia (Anderson et al., 2002c) with reduced blood flow
through the gland demonstrated at minimal secretion rates (Kent and
Olson, 1982). The intermittent nature of rectal gland secretion suggests
that it may be regulated by a series of endocrine/neuroendocrine factors
acting to regulate blood flow. As Ang II is a potent vasoconstrictor in
elasmobranchs (Tierney et al., 1997b; Hamano et al., 1998) and Ang II-like
binding sites and angiotensin converting enzyme like activity have been
reported in the rectal gland of S. canicula (Masini et al., 1994; Hazon et al.,
1997a), Ang II seemed a likely candidate to influence rectal gland
function. However, perfusion of the isolated rectal gland of S. canicula with
109 M Ang II did not affect either secretion rate or vascular perfusion of
the secretory epithelium (Anderson et al., 2002c). Several factors have
been reported to both stimulate (CNP, Solomon et al., 1992; VIP, Stoff
et al., 1977; rectin, Shuttleworth, 1988; scyliorhinin II, Anderson et al.,
1995a) and inhibit (NPY, Silva et al., 1993; somatostatin, Silva et al., 1985)
rectal gland function, and it may be that the actions of Ang II involve
interactions with some of these factors (Anderson et al., 1995b), although

Fish Osmoregulation
it should also be considered that some of the reported stimulatory effects

may be species dependent.
GILL FUNCTION
Gills play an important role in salt balance in teleost fish and
autoradiographic studies suggests the existence of Ang II binding sites
(Table 4.1; Cobb and Brown, 1992) that may have a significant role in
osmoregulation. One of the few studies of the effects of Ang II on gill
function showed a reduced transepithelial potential in the isolated
perfused gill of flounders, suggesting reduced chloride transport (Lyndon,
1993). The idea of Ang II actions on branchial transport processes has
since been supported by the identification of Angiotensin receptors on
chloride cells of the eel (Marsigliante et al., 1996, 1997a). Adaptation to
SW increases the number of chloride cells (and associated Na+, K+ATPase activity) linked to the role of the gills in sodium efflux, so it is
perhaps not surprising that Ang II binding sites in the gills were shown to
increase after acclimation to SW, but even after normalization to the
number of chloride cells present a three fold increase in receptor density
was reported (Marsigliante et al., 1997a), supporting the idea that Ang II
regulates gill function. There are many hormones that influence gill Na+,
K+-ATPase activity (e.g. cortisol, growth hormone, insulin growth factor)
and to the list we must add Ang II. The action of Ang II on gill function
is rapid with short-term exposure of gill homogenates to Ang II or a 30 min
perfusion of gills with Ang II resulting in increased Na+, K+-ATPase
activity in FW-acclimated eels (Marsigliante et al., 1997a). These studies
also showed the existence of two Angiotensin receptor isoforms in the gills
with both present in SW-acclimated eels, but one was lost in the FWacclimated eels, but these complexities that have yet to be fully
understood.
In contrast to teleost fish, the role of the gill in elasmobranch
osmoregulation is poorly understood. Chloride cells have been identified
in elasmobranch gills (Wright, 1973; Laurent and Dunel, 1980), but
elasmobranch branchial Na+-K+-ATPase activity was reported as 10-15
times less than that found in teleosts (Jampol and Epstein, 1970).
Investigation of Na+, K+-ATPase activity and abundance in D. sabina
(Piermarini and Evans, 2000) demonstrated that SW acclimation induced
a decrease in the activity and relative abundance of Na+, K+-ATPase in
the gills. In FW, D. sabina were shown to have a higher number of Na+,
K+-ATPase-rich cells localized primarily on the gill lamellae. The activity,

location and abundance of Na+, K+-ATPase in FW-acclimated D. sabina

suggests a role in ion-uptake for Na+, K+-ATPase-rich cells (Piermarini
and Evans, 2000). Following the identification and localization of the
vacuolar proton-ATPase (V-H+-ATPase) in the gills of D. sabina, a dual
role was proposed for V-H+-ATPase and Na+, K+-ATPase cell types in
combined regulation of sodium and chloride with acid base balance in
both FW and SW (Piermarini and Evans, 2001).
In terms of RAS control of gill function in elasmobranchs, the only
evidence available to date is indirect. Receptors for Ang II have been
identified in membrane fractions prepared from gill cells of the Japanese
dogfish, T. scyllia (Tierney et al., 1997a). However, it is not known whether
Ang II acts on the gill epithelia in a similar way to teleost fish although it
is highly probable that Ang II will influence the perfusion of blood through
the gills due to its vasopressor action on elasmobranchs vasculature
(Hazon et al., 1999).
STEROIDOGENIC ACTIONS OF ANGIOTENSIN AND
LINKS TO OSMOREGULATION
In higher vertebrates, a major role for Ang II is in control of sodium
retention through the stimulation of adrenal steroid release (Kobayashi
and Takei, 1996). There is considerable evidence that a similar action
occurs in teleost fish. For example, Ang II has been shown to stimulate
secretion of cortisol, the major interrenal corticosteroid, after intravenous
administration to flounder in vivo. Plasma cortisol increased over a more
prolonged time course than the elevation in blood pressure (Perrott and
Balment, 1990). Although the dose employed was high (2.5 mg/kg body
wt), the Ang II sequence used in this study (Asp1, Val5-Ang II) is now
known to not be the endogenous sequence and this may have reduced
sensitivity. In trout, Ang II has been shown to stimulate cortisol secretion
in vitro from perfused interrenal tissue acting either synergistically or
additively with adrenocorticotrophin (ACTH) (Decourt and Lahlou,
1987; Arnold Reed et al., 1994). In teleosts, cortisol has well-established
osmoregulatory actions particularly on the gills and intestine, so
corticosteroidogenic actions of Ang II in teleosts could indirectly have
profound osmoregulatory effects.
In the elasmobranch fish, T. scyllia, the greatest specific binding of Ang
II occurred in the interrenal gland (Tierney et al., 1997a) and both
homologous (Anderson et al., 2001b) and heterologous (OToole et al.,
Fish Osmoregulation
1990) Ang II have been shown to be potently steroidogenic in S. canicula.

The major corticosteroid produced by the elasmobranch interrenal gland
is 1a-hydroxycorticosterone (1a-OH-B) (Idler and Truscott, 1966) and
the occurrence of receptors for 1a-OH-B in the rectal gland, gill and
kidney of Raja ocellata (Idler and Kane, 1980) suggests osmoregulatory
roles, but there is much less information on the actions of corticosteroids
in non-teleostean fish. Armour et al. (1993) showed that 1a-OH-B acts on
renal and extra-renal sites to reduce sodium loss, so Ang II can be
predicted to indirectly influence sodium balance. Although the exact
osmoregulatory roles of 1a-OH-B has yet to be determined, it is clear that
the steroid may influence salt and/or urea flux.
In the lungfish, the most closely related of the fishes to the tetrapods,
corticosterone, aldosterone and cortisol have all been reported to be
present in the plasma with increased levels of corticosterone or
aldosterone resulting from injection of Ang II (Joss et al., 1994), but the
effects varied depending on the amino acid sequence of the peptide. Since
this study, native Ang II has been identified in the Australian lungfish (Fig.
4.2; Joss et al., 1999) and this allows comparison of the effects of native and
non-native sequences. Intramuscular injection of Asn1 Val5-Ang II, the
endogenous peptide, increased circulating levels of aldosterone, as did Asp
Ile5-Ang II (Joss et al., 1994). More recently, further studies have
confirmed the release of aldosterone in vitro by perfused interrenal tissue
stimulated with lungfish Ang II, but alteration of the first amino acid from
asparagine to aspartic acid was sufficient to block the release of
aldosterone, whereas isoleucine substitution for valine as the 5th amino
acid restored selectivity, but with a reduced sensitivity (Joss et al., 1999).
Studies to investigate whether aldosterone in lungfish acts on sodium
regulation, as in tetrapods are now needed.
SUMMARY AND FUTURE DIRECTIONS
This chapter has reviewed our understanding of the integrated actions of
the systemic RAS of fish groups and the recent evidence of local paracrine
systems. It is as yet unclear exactly how systemic and local systems operate
alongside each other. Differences in receptor affinities as well as local
concentrations of Ang II or other angiotensin fragments may ultimately
explain how the two systems operate in tandem, but further work is
required to complete the jigsaw. Their roles in regulating drinking, gill and
kidney function and stimulating longer-acting steroidogenesis with further
!
osmoregulatory actions form important elements in contributing to the

achievement of body fluid homeostasis, but much remains to be explored.
Our current knowledge is perhaps greatest amongst the teleosts but
considering that the elasmobranch RAS has only been discovered
relatively recently, much progress has been made in elucidating
physiological roles in the osmoregulatory processes of marine
elasmobranchs. However, there are considerable gaps in our knowledge
and, in particular, relatively little is known of RAS function in the small
number of fully euryhaline elasmobranch fish that inhabit freshwater and
seawater environments, and nothing is known for the members of the ray
family Potamotrygonidae (e.g., Potamotrygon) that are considered to be
permanently resident in freshwaters in the Amazon and Orinoco river
systems, as much as 4500 km from the sea. Although there is evidence of
a RAS in the agnathan lampreys and this system has been shown to be
activated by volume depletion with an apparent interactive effect of
sodium/osmoreceptors, and after an increase in the external osmolality, as
yet our understanding of the role of the RAS in these fish (or whether they
too possess paracrine RASs) is relatively poor. A vasoconstrictor response
is clear and this may represent the fundamental (primitive) action of the
agnathan RAS as a distinct role in stimulation of drinking is not apparent
and the action on the kidney relatively weak, potentially compromised by
vasopressor actions.
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+0)26-4
#
Effect of Water pH and Hardness
on Survival and Growth of
Freshwater Teleosts
Jorge Erick Garcia Parra1 and Bernardo Baldisserotto2, *
INTRODUCTION
The freshwater contains a variable amount of dissolved substances (salts
and organic compounds) (Table 5.1), depending on the soil in which it
occurs. Water acidification may occur in places where the soil contains
acidic cations, as Al3+, or iron pyrite, which under oxygenating
conditions, forms sulfuric acid (Zweig et al., 1999). Some lakes are acidified
by streams with sulfuric and hydrochloric acid of volcanic origin, as Lake
Usoriko (Japan) (Takatsu et al., 2000). The presence of humic and fulvic
acids, formed in the soil through the decomposition of organic matter, can
Authors addresses: 1Departamento de Cincias Agrrias, Universidade Regional Integrada do
Alto Uruguai e das Misses Campus Santiago, 97700.000 Santiago, RS, Brazil.
2
Departamento de Fisiologia e Farmacologia, Universidade Federal de Santa Maria,
97105.900 Santa Maria, RS, Brazil.
*Corresponding author: E-mail: bernardo@smail.ufsm.br
136
Fish Osmoregulation
Table 5.1 Ion concentration (mmol) in some freshwater environments.
pH
Na+
K+
Ca+
Mg+
Cl
SO 4
HCO 3
Hardness
Rio Negro,
Brazil
Nijmejen,
The Netherlands
Pantanal,
Brazil
Lake Usoriko,
Japan 4
Lake Van,
Turkey 5
3.9 -5.0
0.039
0.006
0.007
0.004
0.014
0.003
0.018
0.95
7.6
5.0
0.06
0.8
0.2
4.2
0.5
98.7
10.2
11.1
3.0
0.23
0.04
27.0
3.0-3.6
1.07
0.07
0.27
0.08
1.35
1.34
15.7
9.8
336.9
13.0
0.11
3.9
153.7
24.3
366.5
Mortatti and Probst (2003); Li et al. (1995), Galvo et al. (2003); 4Takatsu et al. (2000); 5 Danulat
and Kempe (1992)
also reduce water pH down to 3.5, as observed in Amazonian blackwaters

(Matsuo and Val, 2003) and some densely vegetated swamps and bogs of
North America (Patrick et al., 1981; Gonzalez, 1996). Alkaline waters may
be the consequence of phytoplankton or aquatic plants blooms, which
decrease the CO2 available in the water during daylight (Wood, 2001).
There are also natural alkaline lakes, some of them with high
concentration of ions (Table 5.1). Soft waters have a low content of salts,
mainly Ca2+ and Mg 2+, but if the soil contains limestone, water can
dissolve large amounts of Ca2+ and Mg2+ salts and is then termed hard
water (Baldisserotto, 2003). In some places, water hardness can also be
increased by the presence of Ba2+ and Fe2+.
SURVIVAL IN ACIDIC AND ALKALINE WATERS
Most teleosts species survive to acute pH changes down to water pH
4.05.0 or up to 9.0-10.0 (Table 5.2) (Alabaster and Lloyd, 1982), but
exposure to more acidic or alkaline waters is lethal within a few hours
(Figs. 5.1 and 5.2). However, species that inhabit acidic waters, as cardinal
tetra, Paracheirodon axelrodi, and banded sunfish, Enneacanthus obesus, can
withstand water pH 3.5 indefinitely (Gonzalez, 1996; Matsuo and Val,
2003). Juveniles and adults of Tribolodon hakonensis survive in the acidic
waters of Lake Usoriko, but must migrate to neutral waters to breed
(Satake et al., 1995). Some species, as the cyprinid Chalcarburnus tarichi,
the Lahontan cutthroat trout (Oncorhynchus clarki henshawi), and the
scale-less carp Gymnocypris przewalskii live in alkaline waters (9.4-9.8), but
137
Table 5.2 Water pH survival range (100% survival) of some freshwater teleosts species.
nd non-determined.
Species
Enneacanthus obesus
Paracheirodon axelrodi
Tribolodon hakonensis
Odontesthes bonariensis
Prochilodus lineatus
Rhamdia quelen
Oncorhynchus mykiss
Alcolapia grahami
lower pH
Higher pH
Exposure
time (days)
3.5
3.5
3.6
4.9
4.0
4.0
4.4
5.0
nd
nd
nd
10.4
9.5
9.0
9.2
11.0
21
5
3
4
5
4
15
5
Reference
Gonzalez (1996)
Gonzalez et al. (1998)
Kaneko et al. (1999)
Gmez (1998)
Zaniboni-Filho et al. (2002)
Zaions and Baldisserotto (2000)
see Alabaster and Lloyd (1982)
Reite et al. (1974)
perform annual spawning migrations into rivers with lower water pH (8.28.5) (Danulat and Selcuk, 1992; Wilkie et al., 1994; Wang et al., 2003). A
species that lives and reproduces in a very alkaline water pH (10.0) is the
Lake Magadi tilapia, Alcolapia grahami (Wilson et al., 2004).
GENERAL ASPECTS OF OSMOREGULATION IN ACIDIC
AND ALKALINE SOFT WATERS
Mortality of fishes exposed to acidic soft water seems related to a decrease
of around 50% of plasma ions, mainly Na+ and Cl (Freda and McDonald,
1988). The rapid ions loss during acid exposure entrain hematological
(increase of hematocrit, hemoglobin and plasma protein) and fluid
volumes disturbances which kill the fish through circulatory failure
(Wood, 1989). Decrease on plasma Ca2+ and Mg2+ was observed in
rainbow trout exposed to water pH 4.0 for 22 days (Giles et al., 1984), but
not in the same species, common shiners (Notropis cornutus), and yellow
perch (Perca flavescens) after 14 days (Freda and McDonald, 1988). In low
water pH, acid load through the gills is the source of acid-base disturbance,
and there is an increase of H+ and NH4+ excretion by the urine to
compensate this problem (Wood et al., 1999).
Exposure to low water pH increases branchial Na+ efflux due to an
opening of tight junctions of gill epithelia, increasing ion loss by a
paracellular route (Gonzalez, 1996; Wood, 2001). There is also a decrease
on Na+ influx when fishes are exposed to acidic waters, with a blockade
of almost 100% at pH 4.0 in rainbow trout. The Na+ uptake blockade is
caused by inhibition of the apical Na+/H+, NH4+ exchangers by external
138
Fish Osmoregulation
Fig. 5.1 Accumulated mortality as a function of time in Prochilodus lineatus (A) and
Rhamdia quelen (B) exposed to acidic water pH and soft water (20-30 mg L1 CaCO3).
Adapted from Zaions and Baldisserotto (2000) and Zaniboni et al. (2002).
H+, or by creating a gradient too step for further extrusion of protons

(Wood, 2001). However, as species that live in very acidic waters are able
to take up salts at water pH 3.5-4.0, it seems that the model proposed for
rainbow trout is not applicable to these species (Gonzalez et al., 1998,
2002).
139
Fig. 5.2 Accumulated mortality as a function of time in Prochilodus lineatus (A) and
Rhamdia quelen (B) exposed to alkaline water pH and soft water (20-30 mg L1 CaCO3).
Adapted from Zaions and Baldisserotto (2000) and Zaniboni et al. (2002).
The main problems in alkaline waters are the inhibition of ammonia

excretion and increase of CO2 excretion (Wood, 2001). At circumneutral
water pH ammonia leaves the gills by diffusion in the form of NH3, and is
converted to NH +
4 in the water, maintaining a favorable gradient for NH3
140
Fish Osmoregulation
diffusion. When the water is alkaline, there is less H+ available to

transform NH3 into NH +
4 , and the NH3 gradient bloodwater decreases.
The corresponding decrease in water CO2 creates a higher bloodwater
gradient, which promotes branchial CO2 losses (Wilkie and Wood, 1996).
The resultant respiratory alkalosis increases plasma pH levels, as observed
in rainbow trout exposed to water pH 10.1 (Yesaki and Iwama, 1992). In
addition, high water pH also inhibits branchial Na+/NH +

4 and Cl /HCO 3
exchangers (Wilkie and Wood, 1996), which explains reductions of plasma
Na+ and Cl in rainbow trout (Yesaki and Iwama, 1992).
Species adapted to very alkaline waters developed mechanisms to
overcome these problems. Lake Magadi tilapia excretes virtually all of its
waste nitrogen as urea (Wilkie and Wood, 1996), and base excretion is
possible through a modified seawater type chloride cell (Laurent et al.,
1995). C. tarichi, from Lake Van, also excretes urea, but NH3 excretion is
maintained, either by the gills due to the specific composition of Lake Van,
which allows maintenance of the gills-water NH3 gradient or by an
increase of renal excretion (Danulat and Kempe, 1992). On the other
hand, Lahontan cutthroat trout survives in alkaline waters because of its
ability to reduce ammonia production and possibly stimulation of gill
Cl/HCO 3 exchange by branchial cell chloride proliferation (Wilkie et al.,
1994). The scale-less carp G. przewalskii also reduces NH3 production,
accumulates this metabolite in the muscle to attenuate levels in other
tissues, and possibly incorporates NH3 into amino acids (but not urea)
(Wang et al., 2003).
EFFECT OF WATER HARDNESS ON SURVIVAL AND
OSMOREGULATION
Rainbow trout maintain constant plasma Ca2+ irrespective of waterborne
Ca2+. Specimens of this species exposed to low waterborne Ca2+
(2.5 mg L1 CaCO3) showed an increase in number of chloride cells on
lamellae and large apical surfaces to increase ion uptake (Perry and Wood,
1985). The armored catfish, Hypostomus tietensis, trara, Hoplias
malabaricus, and jej, Hoplerythrinus unitaeniatus, when transferred from
1.7 mg L1 CaCO3 to distilled or deionized water also showed high
proliferation of gill chloride cells, but the apical surface in direct contact
with the external medium increased only in trara in the first days after
transference (Fernandes and Perna-Martins, 2002; Moron et al., 2003). In
addition, in armored catfish kept in distilled water, the chloride cells were
141
buried and recessed under adjacent pavement cells and the apical surface
was arranged in a sponge-like structure, developing an apical crypt. These
modifications may be an adaptation to provide a microenvironment inside
the crypt, preventing ion loss and favoring ion uptake in a much diluted
environment (Fernandes and Perna-Martins, 2002). Transference of trara
from 1.7 to 85 mg L1 CaCO3 also induced a transient chloride cell
proliferation in the lamellar epithelia, but the same was not observed in
jej (Moron et al., 2003).
Channel catfish, reared at 407 mg L1 CaCO3, accumulates taurine
and other amino acids in the muscle-free pool as a strategy for elevating
osmolality in hard water without raising the ionic concentration of the
body fluids. This strategy is analogous to the accumulation of urea in other
vertebrates, but provides an additional advantage: taurine accumulation is
metabolically safe. However, as the absolute changes in free amino acids
in muscle and plasma were small (compared to fish exposed to 17.9 mg L1
CaCO3), they might play a limited role in the overall osmoregulation of
channel catfish (Buentello and Gatlin, 2002).
Most Mg2+ uptake is by food intake, but if the water presents an
adequate amount of Mg2+, branchial uptake may be enough to
compensate a low-Mg2+ diet in some species (Bijvelds et al., 1998).
However, in Mozambique tilapia, Oreochromis mossambicus, Mg 2+ intake
from the watereither via the integument or drinkingdid not increase
in low-Mg2+ fed fish, despite an increased opercular chloride cell density
(Bijvelds et al., 1996). High waterborne Mg2+ (up to 50 mmol) did not
affect Gasterosteus aculeatus, Mozambique tilapia and goldfish, Carassius
auratus (Bijvelds et al., 1998).
Most teleosts exposed to acidic or alkaline waters showed a higher
survival in hard rather than in soft waters (Freda and McDonald, 1988;
Yesaki and Iwama, 1992; Laitinen and Karttunen, 1994; Townsend and
Baldisserotto, 2001). Rainbow trout exposed to water pH 3.0 or 3.2
presented higher survival at water hardness 165 mg L1 CaCO3 than those
exposed to the same pH and water hardness 10 mg L1 CaCO3 (McDonald
et al., 1980). Specimens of this species transferred from water pH 6.8 to
10.1 showed decreased Na+ and Cl plasma levels at water hardness
4.0 mg L1 CaCO3, but when maintained at water hardness 320 mg L1
CaCO3, did not change ion levels in the plasma and also showed higher
survival at water pH 10.1 (Yesaki and Iwama, 1992). In addition, survival
was higher in recently hatched rainbow trout larvae exposed to pH 4.7 and
142
Fish Osmoregulation
water hardness of 90 mg L1 CaCO3 (increased with addition of Ca2+ and

Mg2+) than in those exposed to the same pH and water hardness of
12 mg L1 CaCO3 (Laitinen and Karttunen, 1994). The increase of water
hardness from 20 up to 300 mg L1 CaCO3 (using CaCl2) increased the
survival rate of silver catfish juveniles exposed water pH 3.75, 10.0 and
10.5, but did not affect significantly survival (which was 100%) at less
extreme pH even at 600 mg L1 CaCO3 (higher values were not tested)
(Townsend and Baldisserotto, 2001).
The protective effect of water hardness against low pH is dependent
of the affinity of branchial tight junctions for Ca2+. This ion is important
at stabilizing these tight junctions and, consequently, decreasing gill ion
loss (Wood, 2001). Apparently, one of the mechanisms for survival of
species that live in very soft waters, as those from the Rio Negro and the
banded sunfish, is the high branchial affinity for Ca2+, because even water
hardness of 0.4-2.0 mg L1 CaCO3 provides enough Ca2+ to saturate tight
junctions binding sites (Gonzalez et al., 1998). However, dissolved organic
carbon present in high amounts in these blackwaters also protects in some
way against the deleterious effect of low water pH (Gonzalez et al., 2002).
Another strategy is used by yellow perch, because waterborne Ca2+ levels
do not affect Na+ uptake in this species. It seems that this species has
transporters with high affinity by Na+ and, therefore, Na+ influx is
maintained even at water pH 4.0 (Freda and McDonald, 1988). However,
for most of the species studied, the increase of water hardness (waterborne
Ca2+) decreases ion loss in acidic pH (Fig. 5.3).
EFFECT OF WATER PH ON HATCHING AND GROWTH
The 6.5-9.0 water pH range is usually considered best for teleosts
reproduction and growth (Zweig et al., 1999), and any increase of acidity
of the water impairs hatching and growth. Gametogenesis is impaired and
11% of the fertilized embryos showed deformation in rainbow trout
exposed to pH 4.5. Hatching does not occur in grumat, Prochilodus
lineatus (Reynalte, 2000), and mortality is high in common carp, Cyprinus
carpio (Jezierka and Witeska, 1995), when fertilization is done at pH 5.0,
and is affected in Perca fluviatilis exposed to pH 5.5 or lower (Runn et al.,
1977). Exposure to low pH (5.5-6.0) also reduced length and weight of
silver catfish larvae compared to those maintained at pH 8.0-8.5 (Lopes
et al., 2001). Similar results were obtained with the flagfish, Jordanella
floridae, whose larvae showed reduced growth when exposed to pH 5.5

0
143
Cl
+
Na
Net flux (mol kg h )
500
1000
1500
2000
2500
3000
10
20
100
Ca
2+
700
1
(mol L )
Fig. 5.3 Whole body ion net fluxes in tambaqui, Colossoma macropomum, exposed to
water pH 3.5 and different waterborne Ca2+ levels. Data from Gonzalez et al. (1998) and
Wood et al. (1998).
compared to larvae maintained in the 6.0 - 6.8 range. In addition, exposure

of larvae of this species to pH 5.0 impaired growth and reduced survival
compared to pH 5.5 (Graig and Baksi, 1977).
Brook trout fingerlings presented lower growth at water pH 5.5, 6.0
and 6.5 than at pH 7.1 (Menendez, 1976), and at water pH 4.2-5.0 than
5.2-6.5 (Norrgren and Degerman, 1992). Rainbow trout showed better
growth at neutral pH (7.2) than at acidic pH (4.4) (Nelson, 1982).
Exposure of silver catfish fingerlings to alkaline (9.0) or acidic water (5.5)
also reduced growth compared to neutral pH (7.5) (Copatti et al., 2005).
However, post larvae of grumat showed better development and survival
at waters pH 6 than at pH 7 and 8 (Reynalte et al., 2000), and exposure
of brown trout, Salmo trutta, to water pH 5.0, 5.44 and 6.0 did not
influence standard growth rate (Norrgren and Degerman, 1992).
Additional studies regarding the growth of fish whose natural
environment is acidic or alkaline (as those described in the previous
sections of this chapter) are still lacking. Probably, their best pH range for
growth is not circumneutral, because the freshwater angelfish,
Pterophyllum scalare, from Rio Negro, showed better growth in the 5-6.9
pH range (Chellappa, 2005).
144
Fish Osmoregulation
EFFECT OF WATER HARDNESS ON HATCHING AND

GROWTH
Water hardening (swelling process of flaccid newly shed eggs when they
first contact water and absorb water) of fertilized eggs varies according to
species and water hardness. When hardness is low, increase in egg
diameter is greater (Gonzal et al., 1987; Spade and Bristow, 1999). Survival
of eye-up eggs of rainbow trout, Atlantic salmon and brook trout is higher
when eggs are hatched at 139-230 mgL1 CaCO3 (Ketola et al., 1988).
Hatching rate of stripped bass, Morone saxatilis, is higher at 200 mgL1
CaCO3 (Spade and Bristow, 1999), and the recommended range for
hatching of silver carp, Hypophthalmichthys molitrix, is 300500 mgL1
CaCO3 (Gonzal et al., 1987). However, higher larval survival and growth
in African catfish, Clarias gariepinus, and silver catfish is in the
60-70 mgL1 CaCO3 water hardness range (Molokwu and Okpokwasili,
2002; Silva et al., 2003, 2005; Townsend et al., 2003) (Fig. 5.4). Channel
catfish swim-up fry exposed to 0, 0.4, 2, 4, or 40 mgL1 Ca2+ (0, 1, 5, 10,
and 100 mgL1 CaCO3) showed best growth at 4 and 40 mgL1 Ca2+
(10 and 100 mgL1 CaCO3) (Tucker and Steeby, 1993). The same authors
observed an abnormal behavior (fry appeared lethargic and were spread
out over the bottom) in water with low Ca2+ concentration (below
2 mgL1 Ca2+). At least in case of silver catfish larvae, the increase of
water hardness from 20 to 70 mgL1 CaCO3 using either Ca2+ or Mg2+
improved hatch rate, but increase of waterborne Ca+2 above 20 mgL1,
irrespective of water hardness, is not recommend for incubation of silver
catfish eggs because it reduced post-hatch (2 days after hatching) survival
and larval weight and length after 21 days (Silva et al., 2003, 2005). On
the other hand, 30-40-day-old striped bass fingerlings presented higher
survival at 278 mgL1 CaCO3 than at 8-10 mgL1 CaCO3, but the
increase of water hardness must be done with CaCl2 and not MgCl2,
showing the importance of Ca2+ in this period (Grizzle et al., 1990).
However, survival of recently hatched larvae (still with yolk sac) of this
species were not affected by water hardness of 3-250 mgL1 of CaCO3,
indicating that the mechanisms for osmoregulation are different than in
older striped bass (Grizzle et al., 1992).
White bass, Morone chrysops, and sunshine bass, M. chrysops female
Morone saxatilis male (23-30-day-old) died within a few hours in water
with 5-6 mgL1 CaCO3. Addition of NaCl (0.7-5.0 gL1) alone did not
increase the survival rate in white bass, and only 25% sunshine bass
145
Fig. 5.4 Survival (A) and length (B) of African catfish (second day after hatching) and
silver catfish (9 days after hatching) larvae to different water hardness levels. Data from
Molokwu and Okpokwasili (2002) and Townsend et al. (2003). Different letters indicate
significant difference among different water hardness in the same species.
survived. Survival rate of white bass increased with 5.0 gNaCl L1 and
increasing water hardness up to 85.7% at 35 mgL1 CaCO3, and a higher
concentration of Ca2+ (up to 50 mgL1 CaCO3) did not increase survival.
Addition of Ca2+ to increase water hardness to 210 mgL1 CaCO3
increased sunshine bass survival to 64% (Grizzle and Mauldin, 1999).
However, neither the hatching rate nor the growth of Mozambique tilapia
larvae were affected by exposure to waters with 2-3 or 88-96 mgL1
CaCO3 (using CaCl2 or CaSO4 to increase water hardness) (Hwang et al.,
1996).
146
Fish Osmoregulation
Water hardness from 12.5 to 200 mgL1 CaCO3 (increased with

CaCl2) did not significantly affect the final weight, food conversion,
condition factor, and plasma Ca2+ levels of sunshine bass after 42 days
(Seals et al., 1994). However, water hardness 200 mgL1 CaCO3 improved
sunshine bass post-harvest survival compared to lower water hardness
(Grizzle et al., 1985). The increase of water hardness with MgSO4 up to
400 mgL1 CaCO3 (with MgSO4) reduced the survival rate of channel
catfish juveniles to 0%, but when CaCO3 was used to increase water
hardness survival was higher (95%) (Perschbacher and Wurts, 1999).
Juvenile brook trout reared at water pH 6.5 and water hardness
100 mgL1 CaCO3 presented a higher growth rate than those reared at the
same pH and water hardness 12.5 mgL1 CaCO3. Exposure of juveniles of
this species to water pH 5.3 reduced growth around 33-38% at both water
hardness (Rodgers, 1984). Apparently, the effect of water hardness on
growth varies according to species and water quality. For some species
(those which in the natural environment are found in hard or moderately
hard water) hard water is needed for good development, while in others it
might ameliorate the deleterious effect of non-optimal conditions.
Acknowledgements
B. Baldisserotto received a CNPq (Conselho
Desenvolvimento TecnolgicoBrazil) research grant.
Nacional
de
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+0)26-4
$
Arginine Vasotocin and Isotocin:
Towards their Role in Fish
Osmoregulation
Ewa Kulczykowska
NEUROPEPTIDES ARGININE VASOTOCIN AND

ISOTOCINGENERAL VIEW
Neuropeptides are defined as peptides synthesized in neurons that play an
important role in transmitting information in the nervous system. The
mechanism of known neuropeptides biosynthesis is similar in general: the
gene of the peptide precursor is first transcribed into m-RNA, which is
then translated into the propeptide. After various modifications (i.e.,
proteolysis of the propeptide), the mature peptide packed into secretory
granules is transported via axonal flow to the nerve terminal where it is
stored and released in response to appropriate stimulus.
That view does fully apply to arginine vasotocin (AVT) and isotocin
(IT), fish neuropeptides synthesized in the hypothalamic magnocellular
Authors address: Department of Genetics and Marine Biotechnology, Institute of Oceanology
of Polish Academy of Sciences, Sopot, Poland.
E-mail: ekulczykowska@iopan.gda.pl
152
Fish Osmoregulation
neurons of the NPO (nucleus preopticus) from where they are transported
to the neurohypophysis for storage and release. The identification of the
neuronal origin of both neurohypophysial peptides and evidence for the
presence of separate hypothalamic neurosecretory neurons producing
AVT and IT in fish have been provided by various methods. These
procedures include immunocytochemistry either alone or combined with
carbocyanine tract tracing and confocal double-color immunofluorescent
microscopy (Goossens et al., 1977; Van den Dungen et al., 1982;
Holmquist and Ekstrm, 1995; Saito et al., 2004). The peptides, closely
related to mammalian vasopressin and oxytocin, containing nine amino
acids residues, have been identified in the hypothalamo-neurohypophysial
system of teleosts by Acher et al. (1961, 1962). As is the case with other
neuropeptides, both AVT and IT are produced as a part of larger precursor
molecule. The vasotocin and isotocin precursor sequences consist of a
signal peptide, hormone and neurophysin (Fig. 6.1). Both pro-vasotocin
and pro-isotocin have elongated carboxyl-terminals with a leucin-rich
segment similar to copeptine-like sequence of vasopressin precursor, but
its glycosylation does not appear to be possible. The polypeptide
neurophysin, cysteine rich and capable of binding the neurohypophysial
hormone, has been shown for the first time in fish by Pickering (1968).
Since the early nineties, the structural organization of pro-vasotocin and
pro-isotocin genes has been described in several fish species: Catostomus
commersoni, Oncorhynchus keta, O. masou, Platichthys flesus, Triakis scyllium,
Neoceratodus forsteri, Danio rerio (Heierhorst et al., 1989, 1990; Suzuki
et al., 1992; Hyodo et al., 1997, 2004; Warne et al., 2000; Unger and
Glasgow, 2003). Prior to secretion, each hormone is stored in secretory
granules in the form of a non-covalent complex with its associated
neurophysin, which is important during formation of the mature
nonapeptide hormone. Release of the complex into the blood by exocytosis
leads to its spontaneous dissociation. In many teleost species, especially
salmonids, a duplication of the nonapeptides genes possessing the different
expression level has been presented (Hiraoka et al., 1993).
The homologous neurohypophysial hormones have been identified in
representatives of all vertebrates systematic groups (Acher, 1995; Bentley,
1998). In general, they are arranged in a five-amino acid ring, joined by a
disulfide bridge and a side chain of three amino acids. So far, at least 12
homologous nonapeptides have been identified among the vertebrates
(Fig. 6.2). Amino acid substitution occurs at the second, third, fourth, or
Ewa Kulczykowska
Fig. 6.1
153
Structure of pro-hormones for pro-vasotocin and pro-isotocin in fish.
eighth position in the molecule. The distribution of these natural analogs

is well-defined: in mammals vasopressin and oxytocin are the main
neurohypophysial hormones, in non-mammalian vertebrates arginine
vasotocin (vasopressin-like peptide) is present together with usually one of
the eight already identified variants of oxytocin-like peptide. There is a
great deal of variability in nonapeptides variants between fish (Acher,
1993; Acher and Chauvet, 1995). Both nonapeptides vasotocin and
isotocin are found in all bony fish except for lungfish possessing mesotocin
instead of isotocin and chondrosteans in which glumitocin, aspargtocin,
asvatocin, phasvatocin or valitocin are recognized besides vasotocin. In
cyclostome fish, however, vasotocin is a sole neurohypophysial
nonapeptide. Since vasotocin is present in all vertebrates, it has been
considered as a precursor molecule for the neurohypophysial hormones
family (Bentley, 1998). It is worth emphasizing that the neurohypophysial
hormones present in one species differ by two amino acid substitutions at
the most, but their biological activities are considerable distinct, i.e.,
vasopressinmammalian well-known antidiuretic hormone and
oxytocinhormone involved in parturition and lactation (Bentley, 1998).
Neuropeptides action is generally determined by their binding to the
specific receptors in the central nervous system (CNS), or in other sites of
the body. In the CNS, neuropeptides play a role of neurotransmitters and/
or neuromodulators, while distributed with the circulatory system they act
as hormones. Just the same, AVT and IT may act, either locally in the
CNS or in the peripheral target organs (Goossens et al., 1977; Van den
Dungen et al., 1982; Acher, 1993; Acher and Chauvet, 1995; Goodson
and Bass, 2000).
It is a well-established fact that the neurohypophysial hormones
receptors belong to the superfamily of guanine nucleotide binding protein
(G-protein)-coupled receptors. The topography of the receptors is typical
154
Fish Osmoregulation
ARGININE VASOTOCIN
Cys - Tyr - Ile - Gln - Asn - Cys - Pro - Arg - Gly - NH2
1
2 3
4
5
6
7
8
9
ARGININE VASOPRESSIN
Cys - Tyr - Phe - Gln - Asn - Cys - Pro -Arg - Gly - NH2
LYSINE VASOPRESSIN
Cys - Tyr - Phe - Gln - Asn - Cys - Pro - Lys - Gly - NH2
PHENYPRESSIN
Cys - Phe - Phe - Gln - Asn - Cys - Pro - Arg - Gly - NH2
ISOTOCIN
Cys - Tyr - Ile - Ser - Asn - Cys - Pro - Ile - Gly - NH2
OXYTOCIN
Cys - Tyr - Ile - Gln - Asn - Cys - Pro - Leu - Gly - NH2
MESOTOCIN
Cys - Tyr - Ile - Gln - Asn - Cys - Pro - Ile - Gly - NH2
VALITOCIN
Cys - Tyr - Ile - Gln - Asn - Cys - Pro - Val - Gly - NH2
GLUMITOCIN
Cys - Tyr - Ile - Ser - Asn - Cys - Pro - Gln - Gly - NH2
ASPARGTOCIN
Cys - Tyr - Ile - Asn - Asn - Cys - Pro - Leu - Gly - NH2
ASVATOCIN
Cys - Tyr - Ile - Asn - Asn - Cys - Pro - Val - Gly - NH2
PHASVATOCIN
Cys - Tyr - Phe - Asn - Asn - Cys - Pro - Val - Gly - NH2
Fig. 6.2
Amino acid sequences of neurohypophysial peptides in vertebrates.
with seven helical transmembrane-spanning domains, four extracellular

domains and three intracellular domains (Bentley, 1998) (Fig. 6.3).
Vasotocin and isotocin receptor transcripts are widely distributed in
different fish organs: brain, pituitary, spleen, lateral line, ovary, bladder,
intestine, liver, heart, gills, kidney and skeletal and smooth muscle,
suggesting a function of nonapeptides there (Mahlmann et al., 1994;
Hausmann et al., 1995). So far, to this authors knowledge, AVT and IT
receptors have been cloned in two fish species, C. commersoni and flounder
(Platichthys flesus) (Mahlmann et al., 1994; Hausmann et al., 1995; Warne,
2001). Cloning of the vasotocin and isotocin receptor genes has revealed
the presence of residues that are conserved among nonapeptide receptors
and have been suggested to contribute to the ligand binding domain
(Hausmann et al., 1996) (Fig. 6.3). There is a growing body of evidence
Ewa Kulczykowska
155
Fig. 6.3 Topography of the arginine vasopressin and isotocin receptors. The receptors are
the members of the G protein-coupled receptor superfamily. The conservative regions
marked in black are probably the sites of interaction with the hormone.
that AVT, which binds to the receptors on the pre-synaptic or postsynaptic membranes in the central nervous system of fish, acts as
neurotransmitter and/or neuromodulator and influences reproductive
physiology and related social behavior (Foran and Bass, 1998; Goodson
and Bass, 2000). Recently, the target neurons for brain action of vasotocin
were identified in newts (Lewis et al., 2004). On the other hand, a number
of studies in fish have demonstrated a role of AVT, when distributed with
circulation, in maintenance of water and electrolytes balance,
cardiovascular activity and regulation of endocrine secretion (Babiker and
Rankin, 1978, 1979; Fryer and Leung, 1982; Acher, 1993; Conklin et al.,
1997).
SIGNALS FOR AVT/IT SYNTHESIS AND RELEASE
Several studies have pointed out that synthesis of teleosts nonapeptides in
hypothalamus and their secretion into circulation in the neurohypophysis
have changed in response to environmental salinity (Maetz and Lahlou,
1974; Haruta et al., 1991; Hyodo and Urano, 1991; Perrott et al., 1991).
In early sixties, it was already observable that neurosecretory material in
hypothalamus and pituitary was reduced after transfer of rainbow trout
(Oncorhynchus mykiss), eel (Anguilla anguilla) and stickleback
(Gasterosteus aculeatus) to a hyperosmotic medium (Fridberg and Olsson,
1959; Holmes and McBean, 1963; Sharratt et al., 1964). Moreover, in
156
Fish Osmoregulation
rainbow trout transferred from FW to SW, Carlson and Holmes (1962) and
Elders (1964) demonstrated the transitory decrease in antidiuretic and
pressor activity of pituitary, which recovered after 6 hours. Novel studies
by Hyodo and Urano (1991) applying an in situ hybridization method have
coincided well with those early results. They have shown in rainbow trout,
that proAVT mRNA level is markedly decreased by transfer of fish from
FW to 80% SW and remains consistently low by day 14. Just after
retransfer of fish to FW, proAVT mRNA level rises to the initial FW value.
A significant decrease in proIT mRNA levels in SW salmonid fish is also
observed but only on day 1 after transfer. The results have suggested that
the acute change of water salinity is an important factor influencing
neurohypophysial hormones synthesis and/or release (Hyodo and Urano,
1991; Urano et al., 1994).
The changes in the pattern of secretion from pituitary and resulting
plasma concentrations of the AVT evoked by alterations in environmental
tonicity was exhibited in the flounder, rainbow trout and carp (Cyprinus
carpio) by Perrott et al. (1991). In the euryhaline flounder and trout, the
higher pituitary AVT in FW-adapted fish was associated with a higher
plasma concentration of the hormone compared with SW-acclimated fish.
However, in the stenohaline carp, there was no difference in plasma AVT
between fish adapted to either FW or 40% SW. On the other hand, the
initial response of the euryhaline flounder to hypotonic challenge involved
a decrease in plasma AVT concentration (Bond et al., 2002) similarly to
that observed in rainbow trout by Kulczykowska (1997). In other
euryhaline fish medaka (Oryzias latipes), the AVT content in the pituitary
temporarily decreased after transfer to SW, thus, suggesting an increase of
AVT release during the first hours of SW adaptation. During readaptation
to FW, however, pituitary content of AVT was elevated within the first
2 hours after transfer, again indicating the pronounced storage of the
hormone (Haruta et al., 1991). In dogfish (Triakis scyllium), marine
elasmobranch utilizing a unique strategy for adaptation to elevated salinity
by accumulation of urea, AVT secretory activity was also enhanced by
transfer to hyperosmotic environment (Hyodo et al., 2004). A teleost
species Rivulus marmoratus, dwellers of brackish waters of tropical
mangrove forests, while exposed to different salinities, reacted by a varying
pattern of vasotocin immunoreactivity in the pituitary and in the preoptic
nucleus (Nrnberger et al., 1996). The response of circulating AVT and IT
to osmotic challenge was also reported in rainbow trout transferred from
Ewa Kulczykowska
157
FW to brackish water (BW): the AVT and IT levels increased significantly

2 hr and 24 hr after transfer, respectively. In FW-transferred fish, both
hormones decreased steadily, achieving the lowest values 2 and 24 hr after
transfer. However, after 10 days of fish adaptation to BW plasma, AVT
concentration decreased below FW value, whereas after 10 days of
acclimatization in FW that increased above BW value (Kulczykowska,
1997). In rainbow trout subjected to acute osmotic stress, a significant
increase in plasma AVT, but not in plasma IT level was observed
(Kulczykowska, 2001). Although it was not clear whether plasma AVT/IT
variations reflect changes in AVT/IT production or secretion rates, or
both, but the hormonal responses to the acute and prolonged changes of
water salinity were evident. Yet both hormones synthesis and release
seemed to be differentially sensitive to plasma osmolality changes
(Kulczykowska, 1997, 2001).
Taken together, it appears that in the euryhaline fish species, the
synthesis, storage and secretion of AVT are sensitive to environmental
stimuli such as exposure to extreme salinities. A question here arises: what
is an internal signal for the neurohypophysial hormone release?
It is a well-established fact in mammals that an increase in plasma
osmolality is the principal physiological stimulus for vasopressin secretion.
Also, severe hypovolemia and/or hypotension are strong signals for this
nonapeptide release (Szczepaska-Sadowska et al., 1983). It is not clear,
however, if similar signals are also engaged in vasotocin and isotocin
secretion in fish. A consistent relationship between plasma AVT
concentration and plasma Na+, Cl, and osmolality was demonstrated in
seawater-adapted flounder (Warne and Balment, 1995). In rainbow trout
transferred between FW and BW, plasma vasotocin concentration also
correlated with plasma osmolality (Kulczykowska, 1997). On the other
hand, although an increase in plasma AVT concentration with increasing
plasma osmolality or sodium concentration was apparent for SW-adapted
flounder, this was not the case in FW-adapted fish (Perrott et al., 1991;
Balment et al., 1993; Harding et al., 1997). Thus, the other factor involved
in controlling of vasotocin secretion, i.e., volemic status of the animal, was
considered. However, the volemic expansion or hemorrhage protocols
applied in chronically cannulated flounder studies failed to demonstrate
that plasma AVT level may be sensitive to volemic status (Warne and
Balment, 1995). Therefore, a rapid increase in plasma osmolality seems to
be a major factor to control circulating AVT level in fish.
158
Fish Osmoregulation
ARGININE VASOTOCIN/ISOTOCIN: TARGETS OF ACTION

AND MECHANISMS
Overview
To have a clearer view of the role of neurohormones in osmoregulation in
fish, it is essential herein to remind the general rules of that process. The
biological structures and regulatory mechanisms participating in
osmoregulation in aquatic and terrestrial vertebrates are different, but they
have also many common features, i.e., neurohypophysial hormones
analogs playing similar regulatory roles (Bentley, 1998). Terrestrial
vertebrates are equipped with kidneys, which are highly specialized in
preserving water and ions. Fish, in general, posses less complicated
mechanism for regulation of ions and water balance, because of easier
access to these elements in water. However, water and ions movements in
freshwater and seawater fish require the regulation in the opposite
direction: marine fish must secrete NaCl and conserve water and
freshwater fish must accumulate salt from environment and eliminate
excess water. Many species use both strategies to maintain electrolytes
balance during a lifetime, i.e., migrating salmon, eel, etc. (Bentley, 1971).
There are three main organs engaged in osmoregulation in fish (in
order of importance): gill, gastrointestinal tract (GIT) and kidney. The
responses of fish to osmotic perturbation are summarized in Fig. 6.4.
Osmoregulatory tissues possess a high level of membrane-bound
enzyme sodium ion-potassium ion-adenosinetriphosphatase activity, i.e.,
Na+- K+- ATPase, sodium pump. The gill is the primary site of active ion
transport responsible for body electrolyte homeostasis in teleosts. In
freshwater teleosts, the gills actively absorb salt and passively intake water
due to the osmotic influx, whereas seawater teleosts actively excrete NaCl.
The branchial chloride cells play an essential role in the seawater
acclimation of euryhaline teleost being responsible for Cl secretion
(Bentley, 1971). The second important organ for water and salt exchange
in fish is the gastrointestinal tract. The GITs role differs in various fish
species. Fish living in fresh water scarcely drink, but the intestine actively
transports sodium from the lumen to the blood. Conversely, drinking and
subsequent absorption of water by the intestine is essential for seawater
fish (Bentley, 1971). The kidney does not have a pivotal role in fish
osmoregulation, but its significance should not be underestimated. Fish are
the only vertebrates with kidneys able to produce urine by glomerular and
Ewa Kulczykowska
159
Fig. 6.4 Physiological responses of fish to different water salinities: potential involvement
of the neurohypophysial neuropeptides. Modified from: Kulczykowska, E. (2002). A review
of multifunctional hormone melatonin and a new hypothesis involving osmoregulation.
Reviews in Fish Biology and Fisheries 11, 321-330.
aglomerular mechanisms defined by Beyenbach (1995) and Renfro (1999).

Glomeruli appear to be particularly suited to the excretion of the large
volumes of water accumulated by fish living in fresh water. The renal
tubules of fish are differentiated from one or two tubular segments to the
complement structure of the vertebrate nephron. Thus, the contribution
of the kidney to extracellular fluid homeostasis is not universal in fish, and
renal function spans the whole spectrum from glomerular filtration to
tubular secretion (for review see: Beyenbach, 1995; Renfro, 1999;
Dantzler, 2003; Nishimura and Fan, 2003). The main function of the
proximal tubule of glomerular kidney in higher vertebrates is to reabsorb
fluid. Surprisingly, the renal proximal tubules of glomerular kidneys in
marine and euryhaline fish, e.g., winter flounder (Pseudopleuronectes
americanus), dogfish shark (Squalus acanthias) and killifish (Fundulus
heteroclitus)) appear to secrete fluids containing Na+ and Cl. Interestingly,
the same phenomenon is observed in the kidney of aglomerular toadfish
(Opsanus tau). In elasmobranchs, the kidney is a major site of urea
retention.
160
Fish Osmoregulation
It is a well-established fact in mammals that neurohypophysial

hormone arginine vasopressin regulates water and ions transport by
epithelia (kidney tubules, skin, bladder) by stimulating adenylate cyclase
via V2-type receptors. Other effects of that hormone (vasoconstriction,
glycogenolysis, platelet aggregation) are mediated by stimulation of
phosphoinositide breakdown and/or calcium mobilization via V1-type
receptors (Bentley, 1998). In non-mammalian vertebratesamong them
fisharginine vasopressin is replaced by arginine vasotocin. If vasotocin
triggers both signaling pathways and fulfils essentially similar functions in
fish as vasopressin does so in mammals is not clear and requires
elucidation. A role of the second neurohypophysial hormone in teleost,
i.e., oxytocin-like isotocin is even more enigmatic.
Are osmoregulatory organs: gill, GIT and kidney the physiological
goals for AVT/IT action? This question will be addressed below.
Gill
The first suggestions that the gill may be a target organ for AVT/IT action
appeared as early as in the sixties. The gills of fish living in seawater are
the site of considerable sodium exchange, with the outflux exceeding the
influx by an amount which is about equivalent to that gained through the
gut (urinary loss is insignificant). In seawater fish, chloride cells secrete
chloride actively into the external water. It was proved that injections of
neurohypophysial peptidesvasotocin and oxytocinfacilitated the
branchial outflux of sodium in flounder transferred from fresh water to
seawater (Motais and Maetz, 1967). In fish living in fresh water, however,
sodium and chloride depletion stimulates the accumulation of sodium by
the gills. It was demonstrated in goldfish that isotocin and, to a lesser
extent vasotocin, while administered, increased the rate of sodium uptake
across the gills (Maetz et al., 1964).
A more updated data will evidently broaden this view. There is no
doubt that an adenylate cyclase in plasma membranes from the teleost gill
is sensitive to fish neurohypophysial peptides. In rainbow trout, AVT and
IT, when administered in concentrations between 1 and 100 pM, have
inhibited both basal and glucagon stimulated activity of the enzyme
(Guibbolini and Lahlou, 1987a). The ability of the inhibition appeared to
be sensitive to the environmental salinity, being especially expressed in
high-salt media (Guibbolini and Lahlou, 1987a). The first evidence for a
saturable, reversible and high-affinity specific binding of AVT by cells
Ewa Kulczykowska
161
isolated from the gills of eels adapted to freshwater (FW) and seawater
(SW) have been provided by means of 125I-labelled AVT binding
(Guibbolini et al., 1988). Further studies conducted on rainbow trout gill
epithelium, using specific neurohypophysial analogues, strongly suggested
the presence of a new type of vasotocin and isotocin receptors designated
as NHf (Guibbolini and Lahlou, 1990), closer to the V1 than to the V2type, with reference to the mammalian model. In experiments with the V1
receptor agonists in rainbow trout gill epithelium, reduction of adenylate
cyclase activity have been more noticeable in SW rather than in FW
(Guibbolini and Lahlou, 1990). Then it has been confirmed that the
effects of fish nonapeptides are mediated by a Gi protein sensitive to
pertussis toxin and guanine nucleotides (Guibbolini and Lahlou, 1992).
Shortly afterwards, the molecular structures of AVT and IT receptors has
been established by Mahlmann et al. (1994) and Hausmann et al. (1995)
in teleost fish C. commersoni. The sequence of AVT and IT receptor has
displayed the similarity to the mammalian V1-type receptor and the
oxytocin receptor, respectively. Mutational analysis has shown that the
different regions of the vasotocin receptor participate in hormone binding
(Hausmann et al., 1996) (Fig. 6.3). Subsequently, an AVT receptor from
the euryhaline flounder has been cloned and its specificity for AVT has
been documented (Warne, 2001). It has been demonstrated that
activation of the receptor is coupled to phospholipase C and the
inositolphosphate/calcium pathway, similarly to that presented in C.
commersoni. Moreover, the flounder receptor has been shown to be more
similar to the mammalian V1 than to the V2-type, in terms of its potent
activation by AVP agonists for V1 receptor subtype and its higher
homology with V1-type receptors (Warne, 2001). The AVT receptor
mRNA expression studies confirm gills as a site of the hormone action in
bony fish (Mahlmann et al., 1994; Warne, 2001).
Although the results pointed out the fish gill as a physiological target
organ for neurohypophysial hormones, the heterogeneity of the branchial
epithelium comprising gill chloride, respiratory, pillar and mucous cells
made it difficult to identify the exact target cells. Only when a detailed
study on primary cultures of gill cells from a marine fish sea bass has been
performed, the respiratory like cells have been shown to be an effective
goals for both neurohypophysial peptides (Guibbolini and Avella, 2003).
It has been demonstrated that both AVT and IT induce a dose-dependent
stimulation of Cl secretion through the epithelium. It is consistent with
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Fish Osmoregulation
the phenomenon of ions excretion through the gills observed in fish living
in seawater. It has been suggested that the physiological effects of AVT
and IT are mediated via two pharmacologically similar, V1-like receptors,
located in gill respiratory-like cells. Probably V1-type receptors are also
present in the chloride cells. Early experiments with isolated gill cells from
SW-adapted eels, showed that AVT is a potent regulator of Clsecretion
in the chloride cells (Guibbolini et al., 1988). Moreover, in rainbow trout
adapted to SW, an increase of gill chloride cells number and a more
pronounced inhibition of adenylate cyclase activity by AVT and IT was
observed, which may indicate the multiplication of the AVT/IT receptors
sites located on the chloride cells during SW adaptation of euryhaline fish
(Guibbolini and Lahlou, 1987a, b). Further, the morphological changes in
the chloride cells of the epithelium in respect to the external salinity,
which correlate strikingly with the AVT-binding parameters, point out to
these cells as the site of AVT action (Guibbolini et al., 1988). Also, recent
studies of the estuarine teleost killifish (Fundulus heteroclitus) suggest that
sodium chloride secretion upregulated on return of the fish to full
seawater, is mediated via arginine vasotocin receptors present in
basolateral membrane of mitochondria-rich cell in gill epithelium
(Marshall, 2003).
Considering the fish gill as a target organ for neurohypophysial
hormones, also the branchial blood vessels should be taken into account
as potential effectors of the hormone action. AVT is known to be a
vasopressor in all major vertebrate groups, among them in fish (Le Mevel
et al., 1993; Bentley, 1998). The vascular action of both neurohypophysial
hormones in the branchial vasculature has been shown in many
experiments (Bennett and Rankin, 1986; Oudit and Butler, 1995; Conklin
et al., 1996, 1997, 1999). In the isolated perfused gills of the European eel,
Bennett and Rankin (1986) demonstrated a vasoconstriction of the
arterio-arterial pathway and a decrease in arterio-arterial flow with no
effect on the arterio-venous component of branchial flow. More recently,
Oudit and Butler (1995) have postulated that AVT-induced
vasoconstriction of arterio-arterial pathway is responsible for increased
branchial shunting in freshwater eel. AVT in physiological concentrations
produced contraction in afferent branchial arteries in holostean gar
(Lepisosteous spp.) (Conklin et al., 1996). In rainbow trout, AVT injection
had a greater impact on branchial resistance than it did on systemic
resistance, but in the isolated perfused gill, the hormone affected both the
Ewa Kulczykowska
163
arterio-arterial and arterio-venous pathways (Conklin et al., 1997). In the

same study, the AVT at low concentrations stimulated constriction in the
arterio-venous pathway and thus increased flow through the alternative
arterio-arterial pathway. On the contrary, the AVT at elevated levels
decreased arterio-arterial flow while increased arterio-venous flow
(Conklin et al., 1997). The alterations in redistribution of blood in gills
may change AVT and IT delivery to the sites of ion exchange in gill
epithelium and influence the ions and water transport via epithelium, as
it was suggested by Maetz and Lahlou (1974).
The magnitude of AVT-stimulated constriction of the isolated efferent
branchial artery in rainbow trouthigh above that of other well-known
vasoconstrictorsindicates a significance of AVT in regulation of gill
vascular resistance, at least in this species. Studies in rainbow trout and eel
have shown that the gill vasculature is far less sensitive to IT than to AVT
(Bennett and Rankin, 1986; Conklin et al., 1999). Vascular actions of
neurohypophysial peptides have also been studied in free-swimming,
chronically cannulated flounder by Warne and Balment (1997a, b). The
initial fall in dorsal aortic blood pressure observed immediately after AVT
and IT injections have been proposed to be due to AVT/IT dependent
branchial vasoconstriction. It has been suggested that the AVT primary
effect is a constriction of the arterio-arterial pathway (Warne and
Balment, 1997a, b), as it has been reported in the in vitro perfused gill
preparation by Bennett and Rankin (1986). A subsequent rise in the postbranchial blood pressure in response to AVT and lack of this effect
following IT injection would suggest the presence of different types of
hormones receptors (Warne and Balment, 1997a, b). However, it should
be stressed here that the most of vascular effects in gill have been observed
at hormones doses high above those considered as physiologically relevant.
Also in the study of conscious, chronically cannulated Atlantic cod
(Gadus morhua), the biphasic vascular reaction to administered AVT was
demonstrated, but the dose of the hormone can be considered evidently
as pharmacological. The absence of any response to IT injection was
obvious in this study (Kulczykowska, 1998).
Taking together, a physiological action of both peptides in the regional
blood flow distribution in fish gill and significance of this mechanism in
osmoregulatory process needs to be reconsidered.
164
Fish Osmoregulation
Gastrointestinal Tract (GIT)

It is well known that fish living in freshwater drink little water, but teleosts
in the sea must drink salt water permanently in order to prevent
dehydration. The intestine of freshwater fish actively absorbs sodium from
the food, while fasting the fish gain little sodium from the water they drink.
In seawater, a salt absorption and coupled to this water absorption take
place in teleost fish, but the cartilaginous fish have developed an original
urea-based osmoregulation model and use a rectal salt gland for sodium
chloride excretion (Bentley, 1971).
There are no existing datato this authors knowledgeon the
potential physiological action of neurohypophysial hormones in GIT of
fish, although the IT receptor transcripts in the intestine of C. commersoni
(Hausmann et al., 1995) and AVT receptors in the GITs smooth muscle
of rainbow trout (Conklin et al., 1999) were reported. AVT has been
shown to contract gastrointestinal tissue, but at concentrations 10-100
times higher than those that contract blood vessels (Conklin et al., 1999).
It may mean that GIT is not a natural site of AVT action or else a local
supplementing production of AVT takes place here, as has been shown for
mammalian AVP (Friedmann et al., 1993). An immuno-histochemical
localization of AVP in cells of mucosal epithelium and in fibers near the
capillaries situated along the basal side of the epithelium cells suggests an
action of this nonapeptide in mammalian GIT (Sanchez-Franco et al.,
1986). Whether there is the case of AVT in fish, is a matter of speculation.
In marine cartilaginous fish, the rectal salt gland devoted to sodium
chloride excretion is considered as a potential site of AVT and oxytocinlike peptides action (Acher et al., 1999), but so far, to the authors
knowledge, no investigations have been carried out.
Kidney
A renal function in fish depends on the external environment and can
exemplify hyperosmoregulation in FW medium or hypoosmoregulation in
SW medium. In freshwater teleosts, the major role of the kidney is to
eliminate excess water and to retain electrolytes. In the distal tubule, NaCl
is actively reabsorbed without the accompaniment of water. In marine
teleost, however, the role of the kidney is different, i.e., to conserve water.
The proximal tubules reabsorb NaCl to restore water, but the distal tubules
are degenerated and a considerable volume of water is lost (Bentley, 1971).
Ewa Kulczykowska
165
Adaptations of euryhaline teleosts to both hypo- and hyperosmotic media

probably include changes of both: the glomerular filtration rate and the
permeability of the renal tubules. However, in contrast to welldocumented AVP renal effects in mammals, our current knowledge of
AVT actions in fish kidney are far behind.
In the early studies in chronically cannulated eels, AVT
administration was shown to induce dose dependent changes in urine
production (Chester Jones et al., 1969; Henderson and Wales, 1974;
Babiker and Rankin, 1978). In the freshwater eel, doses less than 0.1 ng/
kg body weight were antidiuretic while doses greater than 1 ng/kg body
weight were diuretic (Henderson and Wales, 1974). Similar results were
obtained by Babiker and Rankin (1978) with low doses of AVT and IT
(1 pg - 1 ng/kg body weight) reducing urine production in eels adapted to
FW (but not in SW fish), and high doses (more than 10 ng/kg body
weight) resulting always in diuresis. In the light of more recently developed
AVT/IT assays (Warne et al., 1994; Pierson et al., 1995; Gozdowska and
Kulczykowska, 2004), sensitive enough to measure the circulating
hormones levels, it appears that the diuretic actions reported in earlier
studies were present only at high, pharmacological hormones
concentrations. Smaller doses of the hormones, which gave plasma AVT/
IT concentrations within physiological range, were antidiuretic.
By analogy with mammalian AVP, two components of AVT/IT action
in fish kidney, i.e., vascular and tubular should be considered. The diuresis
observed after pharmacological AVT doses (100 and more times AVT
physiological levels) was accompanied by a significant increase in systemic
blood pressure (Henderson and Wales, 1974). In many teleost fishin
contrast to mammalsglomerulotubular balance seems to be poorly
developed, and GFR is readily increased by an increase in renal perfusion
pressure (Nishimura and Bailey, 1982). Therefore, the changes in systemic
blood pressure affect glomerular filtration in fish kidney. Thus, AVT and
IT inducing hypertension may be responsible for an increase in GFR, and
in this way affect the water/ions balance in fish. The model of in situ trout
kidney perfused under constant pressure was employed by Amer and
Brown (1995) to evaluate the effect of 109 and 1011 M AVT on
glomerular function. The investigations have clearly demonstrated that
both concentrations of AVT have a potent glomerular antidiuretic action
resulting from the decrease in the filtering population of glomeruli. The
AVT induced antidiuresis accompanied by a fall in the number of filtering
166
Fish Osmoregulation
nephrons has also been shown in freshwater eel (Henderson and Wales,
1974). Moreover, in a model of trout trunk preparation, the renal response
to AVT would appear to be a result of glomerular action of the hormone
rather than tubular one (Warne et al., 2002). It has been proposed by
Warne et al. (2002) that the mechanism of that involved smooth muscle
constriction of the renal arterioles regulating blood flow to the glomeruli.
Whether it would be V1 type AVT receptor, as suggested by Pang et al.
(1983) using mammalian V1 receptor antagonists in the trout trunk
model, still remains to be elucidated. A decrease in GFR, population of
filtering nephrons and urine flow rate induced by AVT has also been
shown in a perfused trunk preparation of Scyliorhinus canicula by Wells
et al. (2002).
In addition to the glomerular effect of the hormones, their tubular
action has also been taken into account. In isolated nephron of the trout,
a dose-dependent rise in cAMP production as a result of the
administration of 105 - 1011 M AVT was demonstrated by Perrott et al.
(1993) and the presence of a receptor similar to mammalian V2-type
receptor was suggested (Balment et al., 1993). More recently, significant
stimulation of intracellular accumulation of cAMP by AVT at lower
concentrations (1012 M) has been observed in the case of in vitro
preparation of trout kidney tubules (Warne et al., 2002). Amer and Brown
(1995) in their trout trunk studies have presented an increase of tubular
reabsorption of water after small dose of AVT (1011 M) and a decrease of
that after the higher dose of the hormone (109 M). On the other hand,
Nishimura et al. (1983) in early studies of the isolated renal tubules in
freshwater trout suggested that in the distal tubule water flux and osmotic
water permeability were not affected by neurohypophysial peptides. As yet,
there has been no clear picture of either a site of AVT/IT action or a
presence of well-defined type AVT/IT receptors in fish renal tubules. The
fish nephron lacks the loop of Henle and cannot produce concentrated
urine by way of water withdrawal in the collecting duct. Thus, the tubular
action of AVT, if any, is probably different from that of AVP in mammalian
kidney and any analogies between both nonapeptides should be drawn
carefully.
The fluid produced by the kidney passes to a urinary bladder, organ of
a high permeability to water and solute, where the composition of renal
output can be modified probably by the hormones. In the 1990s, the AVT
and IT receptors have been identified in urinary bladder in C. commersoni
Ewa Kulczykowska
167
by Mahlmann et al. (1994) and Hausmann et al. (1995), but a direct role
of nonapeptides remains to be determined.
INTERACTIONS WITH OTHER HORMONES
The hormonal regulation of water and ion homeostasis requires
participation and interaction of many endocrine systems at many
functional levels in the organism. Hence, the role of potential relationships
between AVT/IT and other hormones systems contributing to
osmoregulation in fish is considered. However, such interactions, with few
exceptions, have not been studied to date in fish and, therefore, data
presented herein are scarce.
Cortisol, prolactin and growth hormone (GH) have been well
documented to be involved in osmoregulation in fish since the sixties
(Bentley, 1971). The investigations undertaken in goldfish demonstrated
that both AVT and IT can stimulate cortisol secretion in this species and
suggested the corticotropin-releasing factor (CRF) activity of these
hormones (Fryer and Leung, 1982). Moreover, Pierson et al. (1995)
showed that both neurohypophysial hormones induced in a dosedependent way the corticotropin (ACTH) release from trout pituitary. On
the other hand, in view of papers by Fryer et al. (1985) and Olivereau and
Olivereau (1990), the participation of AVT in stimulation of ACTH
release in teleosts seems to be less clear, but can not be excluded. In terms
of other osmoregulatory hormones, i.e., GH and prolactin, there is a lack
of satisfactory data on the relationship with neurohypophysial
nonapeptides, although the binding sites for AVT found in GH-producing
cells in the pituitary may suggest an involvement of AVT in GH release
(Moons et al., 1989).
The renin-angiotensin system (RAS) appears to be an important
factor in the regulation of blood pressure and drinking, and thus in the
control of body fluid homeostasis in teleost fish (Russell et al., 2001).
Hence, a possible interaction between angiotensin II, a potent dipsogenic
hormone, and neurohypophysial hormones in fish has also been
considered. In mammals, administration of angiotensin II stimulates
vasopressin secretion from pituitary (Yamaguchi et al., 1985). In the
chronically cannulated flounder model, however, an inhibitory effect of
angiotensin II on AVT release has been observed (Balment et al., 2003).
It is not yet clear whether angiotensin II is a factor engaged in physiological
control of AVT release in fish, but a potential relationship between
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Fish Osmoregulation
hormones would be of special significance in species migrating from freshto seawater, when angiotensin II stimulate drinking (Takei, 2000).
ANALOGY BETWEEN NEUROHYPOPHYSIAL HORMONES
IN FISH AND MAMMALS: A USEFUL PARADIGM?
The advanced knowledge of AVP engagement in mammalian
osmoregulation and the absence of equivalent data on AVT and IT in fish
are templates for drawing analogies. Is that legitimate?
In mammals, the kidney is a sole important osmoregulatory organ and
vasopressin is a main antidiuretic hormone acting here. On the basis of
functional and pharmacological criteria, two types of renal vasopressin
receptors have been distinguished: V1 receptors which activation is
associated with mobilizing intracellular calcium or stimulating
phosphoinositide breakdown and V2 receptors leading to the activation of
adenylate cyclase. Generally, V1 type receptor is associated with the
vascular action of AVP and V2 type receptor is linked to the tubular
antidiuretic action of the hormone in the distal parts of nephron.
In fish, on the other hand, the many structures such as gill, GIT, salt
gland and urinary bladder act in concert with the kidney so as to maintain
homeostasis of body fluids and electrolytes. Moreover, the kidney seems to
be less important organ in fish osmoregulation process. The precise
characterization of AVT and IT receptors and their distribution in
osmoregulatory organs is far from being established. In fish species studied
to date, i.e., flounder and C. commersoni, AVT receptors have a higher
homology with vasopressin V1 type than with the V2 type and are linked
to the phospholipase C-phosphatidylinositol signaling pathway. On the
other hand, the isotocin receptors are closely related to mammalian
oxytocin receptors and vasopressin V1 type. Also, V2 type receptors linked
to adenylate cyclase seem to be present in fish kidney. A dose-dependent
increase of cAMP production after AVT administration was shown in
trout renal tubules in vitro (Perrott et al., 1993). Moreover, a significant
accumulation of cAMP by AVT within the range of physiological
concentration was presented in trout nephron suspension (Warne et al.,
2002). The occurrence of V2 type receptors was also suggested in other
transport epithelia in fish. Urea excretion in marine toadfish (Opsanus
beta) gill was proposed to involve a specific transport mechanism
analogous to the vasopressin-sensitive renal urea transporter of mammals
linked to V2 type receptors (Walsh, 1997). It was shown in this
Ewa Kulczykowska
169
aglomerular fish, that pulsatile branchial urea excretion was under the
control of arginine vasotocin (Perry et al., 1998). However, the recent
experimental data by Wood et al. (2001) do not confirm this hypothesis.
Therefore, further studies are essential to examine whether in fish nephron
the AVT receptors related to mammalian V2 type are present. The
character of the tubular action of AVT in fish, if any, would be probably
different from that of AVP in mammals, because the fish nephron lacks the
loop of Henle and cannot produce concentrated urine by water withdrawal
in the collecting duct.
Thus, the mammalian paradigm offered to elucidate the mode of
involvement of AVT/IT in osmoregulation in fish, although essential
considering a lack of satisfactory fish data, can be applied only within strict
limit.
SUMMARY. ARE THERE CONVINCING EVIDENCES ON
A ROLE OF AVT/IT IN FISH OSMOREGULATION?
In this chapter, the current state of the knowledge of the role of
neurohypophysial hormones arginine vasotocin and isotocin in fish
osmoregulation has been summarized.
It has been shown that the synthesis of nonapeptides in hypothalamus
and their secretion into circulation from the neurohypophysis are sensitive
to an important environmental factor: salinity. Rapid transfer of teleost
fish between fresh and seawaters results in altered mRNA expression of
AVT precursors in hypothalamic neurons and consequently in altered
content of AVT in pituitary. The patterns of plasma AVT and IT
concentrations in response to external salinity changes suggest the role of
the hormones in the mechanism of fast adaptation. AVT and IT releases
appear to be controlled independently. There are three main
osmoregulatory organs in fish: gill, GIT and kidney, which are considered
as potential goals for neurohypophysial nonapetides action. Physiological
responses of fish to different water salinities and suggested involvement of
AVT/IT are summarized in Fig. 6.4.
There are clearly many unresolved questions to be explored. Data on
the osmoregulatory role of AVT in fish still remains fragmentary.
Implication of isotocin in this process is even less clear. Moreover, the
physiological role of neurohypophysial hormones in the maintenance of
water/ions homeostasis in fish does not seem to be uniform among species,
in contrast to their antidiuretic and sodium-retaining function in
170
Fish Osmoregulation
tetrapods. A mammalian paradigm, although helpful in interpretation of

fish data, needs to be verified by studies in fish. The new experimental
approaches and modern techniques addressed to arginine vasotocin and
isotocin gives a promise to elucidate a role of both nonapeptides in fish
osmoregulation.
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+0)26-4
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Cellular and Molecular
Approaches to the Investigation
of Piscine Osmoregulation:
Current and Future Perspectives
Chris N. Glover
The maintenance of salt and water balance in fish is the consequence of

a tightly regulated, integrated network of molecular and cellular processes
operating within a variety of cell types and across a range of tissues. In
essence, these same cells and tissues, often utilizing the same molecular
and cellular pathways, are capable of ionic and osmotic homeostasis in
environments as disparate as dilute freshwater, where the fish is faced with
salt loss and water gain, and seawater where water loss and salt loading are
the major challenges. Cellular and molecular components of these
Authors address: SCION, Te Papa Tipu Innovation Park, 49 Sala Street, Private Bag 3020,
Rotorua, New Zealand.
E-mail: Chris.Glover@scionresearch.com
178
Fish Osmoregulation
homeostatic pathways are under the control of a complex assortment of

endocrine factors, the presence of which depends on environmental,
developmental and physiological cues. Osmoregulatory hormones may
have distinct effects, depending on circulating concentrations, and their
impact may vary with the presence of other circulating mediators, as well
as the molecular and cellular wiring of the target cell. This wiring itself can
be determined by genetic constitution, life history and environmental
factors. Consequently, even closely related species will have a different
assortment and/or arrangement of osmoregulating machinery and
mediators. Given its complexity, our understanding of exactly how the
processes underlying the ability of fish to maintain homeostasis are
initiated, regulated and integrated is in its infancy. Recent developments
in cellular and molecular techniques will provide the impetus for many of
the advances in this field in future years.
This chapter seeks to provide insight into how advances in cellular
and molecular techniques have been, and can be, applied to further our
understanding of fish osmoregulation. It is not intended to act as a
practical guide to these techniques; nor is it the intent to provide a
comprehensive summary of the progress that has been made. The
emphasis of this review will be on developments in the last decade. Thus,
to a limited degree, it may act as an update to the monograph on cellular
and molecular approaches in fish osmoregulation that appeared ten years
ago (Wood and Shuttleworth, 1995). Many of the techniques and their
applications have changed little over this period and consequently will not
be detailed in this synopsis. This extends to preparations such as the
killifish opercular epithelium, and the accompanying suite of
electrophysiological techniques, which continue to provide important
insights into the passage of ions across osmoregulatory epithelia (e.g.,
Evans et al., 2004; Marshall et al., 2005), but which, in terms of utility and
applicability, remain relatively unchanged. Devoted discussions of the
elasmobranch rectal gland (Silva et al., 1990), and fish urinary bladder
(Marshall, 1995), have been excluded for similar reasons. As a guide, the
material herein will be aimed at the reader with sound background
knowledge of fish osmoregulation, and an understanding of the basic
concepts of cellular and molecular biology.
Chris N. Glover
179
UTILIZATION AND APPLICATION OF CELLULAR

TECHNIQUES IN FISH OSMOREGULATION
As the biological barrier between the internal and external milieux,
epithelia are critical for the maintenance of salt and water balance. In
addition to functions related to ion and water transport, they may also be
involved in essential biological processes such as digestion, gas exchange,
and excretion. By virtue of their tightly regulated multifunctional roles,
epithelia are characterized by both the heterogeneity of their cellular
composition, and their responsiveness to endocrine and neural control.
For discerning mechanisms of physiological action these characteristics are
disadvantageous. This has led to the development of models where
physiological function can be assessed in relatively homogenous cell or
membrane populations free from the confounding actions of central
mediators. The extrication of cells from an environment where they are
exposed to a number of simultaneous inputs, and their subsequent
placement in conditions where they can respond normally but are under
the strict control of the experimenter, permits the detailed elucidation of
molecular, biochemical and physiological pathways. Delineating
mechanisms of ionic and osmotic homeostasis in a relatively simplistic
setting allows the development of hypotheses that can subsequently be
tested in more biologically complicated systems, and across a range of cell
types, tissues and species.
Cellular approaches not only offer scientific advantages, but also
technical, ethical and economic benefits over the use of whole animal
physiological investigations (Castao et al., 2003). Many species of interest
are not easily maintained in laboratory settings, and therefore are not
readily accessible for experimentation. Access to cell lines, for example,
can overcome this problem. The development and use of suitable fish
cellular models will reduce the dependence on whole animal studies, of
obvious benefit from an animal welfare perspective. The possibility of
minimizing expensive animal husbandry, and the ability to utilize a single
animal for multiple manipulations, is of obvious financial benefit.
Cellular techniques utilized in fish biologyand that will be discussed
in the following chapterinclude reconstituted epithelia, single cell
preparations, and isolated cellular components. In all cases, these
techniques facilitate the study of fish osmoregulation by improving
manipulability, homogeneity, and accessibility to the epithelial cells of
greatest interest to the researcher. The utility and applicability of these
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methods will be critically assessed with particular regard to fish

osmoregulation by highlighting the progress made using these techniques
to date, and speculating on how they may advance the field in the future.
Methods often associated with cellular models (i.e., pharmacological
agents) will also be considered.
Yolk-ballsA Novel Surrogate Preparation for Chloride
Cell Investigation
The osmotic challenges faced by early life stages of teleost fish are similar
to those of juvenile and adult forms. However, unlike in their more
developed brethren, the structures primarily associated with osmotic and
ionic control (gill, gut and kidney), are not fully functional (Alderice,
1988; Varsamos et al., 2005). The timing of gill, kidney and gut
development varies among species. However, the mechanism used by
embryonic and larval forms to compensate for passive ion and water flux
appears to be highly conserved. The occurrence of chloride cells on the
integument has been noted in all studied teleost species (e.g., Hwang and
Hirano, 1985, Katoh et al., 2000, Varsamos et al., 2002), and are believed
to be critical for osmoregulation in developing fish. These mitochondriarich cells, characterized by extensive basolateral invaginations, a high
metabolic activity, and a morphology that can change considerably with
osmotic environment, are believed to be important osmoregulatory loci
(Evans et al., 1999). In most developing species, integument chloride cell
clusters are primarily associated with the yolk-sac membrane.
Recently, a method has been developed that permits the separation of
the yolk-sac from the developing embryo, and which may provide an in
vitro model of considerable utility (Shiraishi et al., 2001; Kaneko et al.,
2002). This technique involves the surgical removal of the yolk sac from
the developing fish, in this case tilapia (Oreochromis mossambicus). The
incision wound heals rapidly such that three hours after the surgery, the
yolk sac membrane has completely resealed and encapsulates the yolk
(Shiraishi et al., 2001). This yolk-ball preparation offers many potential
advantages as a tool for examining osmoregulatory function in fish. A
major benefit is that the yolk-sac membrane chloride cells appear to be
similar to those that later develop in the adult gill (Kaneko et al., 2002).
They are mitochondria-rich and immunoreactive to the basolateral
sodium pump (Na+/K+-ATPase) thought to drive ion uptake (Kaneko
et al., 2002). Morphologically, the extensive tubular elongations of
Chris N. Glover
181
branchial chloride cells are replaced by a more compact, flattened

appearance in the yolk sac, a limitation likely imposed by the reduced
thickness of the epidermal layer (Kaneko et al., 2002). Functionally, these
cells appear to respond to changes in environmental salinity in a similar
manner to the adult forms, even in this liberated state. For example, in
response to seawater, cells increase in size and develop characteristic
membrane elaborations (Kaneko et al., 2002). This suggests that the yolk
ball has an inherent mechanism for both sensing and responding to
external salinity. Structural confirmation of this ability was provided by the
demonstration of functional receptors for the important osmoregulatory
hormones prolactin and cortisol in yolk-sac membranes (Shiraishi et al.,
1999).
The yolk ball offers several advantages over the isolated yolk sac
membrane in terms of its usefulness as a model system for the examination
of osmoregulation. In order to maintain a chloride cell population, yolksac membranes required the application of exogenous cortisol, the
hormone closely associated with seawater acclimation (Ayson et al., 1995).
Exogenous application of cortisol had little effect on the responses of
freshwater- or seawater-exposed yolk-balls (Shiraishi et al., 2001).
However, on exposure to seawater, the yolk ball chloride cells responded
both morphologically and physiologically, in a manner similar to that
observed on seawater exposure in vivo (Shiraishi et al., 2001; Hiroi et al.,
2005). Such a response is associated with a cortisol surge (McCormick,
2001). This is an important observation. First, it indicates that the yolkball preparation is capable of sensing osmotic changes in the environment,
independent of higher control. Second, it suggests that the yolk itself may
play a vital role in the osmoregulatory capacity of the preparation. The
ability to respond to seawater exposure is likely to be the consequence of
maternally supplied cortisol present in the yolk (Tagawa, 1996). This is of
particular interest in that it indicates the yolk may play a role
analogous to the circulation in intact animals. Consequently, from an
experimental perspective, it provides a situation whereby the yolk
contents may be manipulated in order to investigate aspects of
osmoregulatory control of chloride cell function. Due to the rapid healing
of the membrane following surgery, it may be possible to introduce salinity
changes and endocrine factors into the yolk, equivalent to infusion or
injection in vivo, but without the side effects of higher control (e.g.,
alterations in blood perfusion, release of endocrine regulators). The yolkball preparation, therefore, allows chloride cell structure and function to
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Fish Osmoregulation
be examined under conditions where both apical and basolateral

influences can be monitored either independently or simultaneously.
Many of the advantages of this preparation are similar to those offered
by double-seeded primary gill cell culture techniques (see below). Both
include populations of chloride cells, and the capacity to control apical
and basolateral exposure conditions. A distinct benefit of the yolk-ball
technique over primary cell cultures as a model system for the examination
of chloride cell function is its relative ease. They are relatively simple to
acquire, maintain viability with a minimum of effort, and can be easily
experimentally manipulated. In contrast, cell cultures are an often
laborious and expensive method.
The yolk-ball preparation is a relatively new development, and there
is much scope for further investigation. To date, the technique has only
been utilized in tilapia. Expansion to other fish species should be
amenable. The manipulability, viability and in vitro nature of the yolk-ball
preparation are of significant advantage in that osmotic environment can
be altered, while systemic concerns are largely eliminated. Thus, the
comparison of yolk-balls between stenohaline freshwater and marine
species, along with euryhaline teleosts, may offer insight into the wiring
of homeostatic control. It is also a method applicable to the investigation
of osmoregulatory development. Yolk balls of different stages may offer
perspectives into the ontogeny of important cellular sensors of, and
responses to, osmotic and ionic imbalance.
Cultured Fish CellsReconstituting Epithelia for
Transport Studies
Branchial cell culture
Investigations of ionic and osmotic regulation in fish generally focus on the
gill (see Evans et al., 1999; Marshall, 2002 for reviews). Its elegant system
of arches, filaments and lamellae result in a branchial surface area that can
represent more than half of the total body surface area. This large surface
is well vascularized with a short diffusional distance between blood and
water. These properties, in association with the often large osmotic and
ionic gradients between the animal and the external milieu, render the gill
a critical osmoregulatory organ. The many other functions of branchial
tissue, including gas exchange and excretion, are also favored by this
morphology, and must be balanced with osmoregulatory demands. The
Chris N. Glover
183
intricate architecture of the gill, combined with its diverse physiological

roles, has been a limiting factor in determining aspects of ion and water
passage across this epithelial surface. The desire for a cellular method that
permits the maintenance of gill properties and function, while offering a
lamellar surface amenable to apical and basolateral manipulation, has
resulted in the development of branchial cell culture methods (Leguen
et al., 2001; Wood et al., 2002). Opercular epithelial preparations of the
killifish incorporate many of these advantages (see Marshall, 1995), but to
date these methods are most useful only as a model for ion transport in
seawater fish, especially with regard to chloride secretion and calcium
uptake (Zadunaisky, 1984; Marshall, 1995; Wood et al., 2002).
Following the successful culture of branchial cells in flasks (Avella
et al., 1994), methods were developedin both marine and freshwater fish
speciesto allow growth of branchial cells on permeable filters (Avella
and Ehrenfeld, 1997; Wood and Prt, 1997). These techniques resulted in
homogenous cultures of pavement cells that formed tight epithelia and
developed apical and basolateral morphologies. Pavement cells are the less
differentiated branchial epithelial cells with dual roles in gas exchange and
ionic regulation (Evans et al., 1999). Mucus-secreting cells and chloride
cells were virtually absent. These cultures permitted analysis of ion
transport characteristics of pavement cells in both marine and freshwater
gills. For example, electrogenic chloride movement was determined using
Ussing techniques in the sea bass, Dicentrarchus labrax (Avella et al., 1999).
The epithelial integrity of these preparations is such that the apical
bathing medium can be replaced with freshwater to recreate conditions
that may exist in vivo.
These single-seeded preparations lack chloride cells, and thus are not
good surrogate models of intact gill. This led to the development of
double-seeded techniques for freshwater rainbow trout. These utilize an
initially cultured layer of pavement cells as a seeding platform for a second
application of gill cells and result in a culture containing chloride cells
(Fletcher et al., 2000). In fact, the relative proportions of cells in these
double-seeded reconstituted cultures resemble those found in freshwater
in vivo (~85% pavement cells, ~15% chloride cells; Perry and Walsh,
1989). These preparations form very tight epithelia and have been used to
reinvestigate the mechanisms of ammonia excretion across branchial
surfaces (Wood et al., 2002), for example. This technique has yet to be
extended to marine fish.
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Fish Osmoregulation
There is a major caveat regarding the usefulness of these preparations

for studies investigating piscine osmoregulation. To date, these cultures
display somewhat anomalous active ion transport properties in freshwater.
Diffusive ion fluxes appeared to closely resemble those of intact fish (Wood
and Prt, 1997). However, in single-seeded preparations lacking chloride
cells, a small active chloride influx was present under asymmetric culture
conditions (basolateral culture medium/apical freshwater). This is
contrary to the established ion transport dogma, which localizes this
process to chloride cells. The active transport component persisted in
double-seeded cultures (with chloride cells), but at a magnitude that was
considerably less than expected, and which represented only a small
proportion of diffusive ion movement (Wood et al., 2002). In doubleseeded cultures, active calcium uptake was present and quantitatively
resembles that in vivo, but was accompanied by an unanticipated active
calcium efflux (Wood et al., 2002).
A recent study suggested a possible reason for observed discrepancies
between cultured cells and the in vivo condition. To mimic circulating
cortisol levels, this hormone was applied to the serosal surface of doubleseeded rainbow trout cultures exposed to apical freshwater (Zhou et al.,
2003). This resulted in the development of a small active influx
component for both sodium and chloride. Although the transport rates
were small, this represented the first demonstration of active sodium
transport by a branchial gill culture. This suggests that hormonal
supplementation of media may provide the key to establishing a
functional, ion transporting cultured gill epithelium. Until active transport
can be established in these models, they have limited utility for many
applications of relevance to the study of fish osmoregulation.
Depending on the nature of the experiment, culturing cells on
supports may offer little advantage over suspended cell techniques
(Sandbacka et al., 1999), or simple gill filament culture systems (Bury et al.,
1998; Mazon et al., 2004). Although lacking cellular polarity, and/or
exhibiting reduced viability, these latter methods are advantageous in
terms of cost and labor. For example, results from studies examining the
impact of hypotonic shock on cells in culture resembled those conducted
with freshly suspended cells (Leguen and Prunet, 2004). This highlights
the integrity of the branchial culture technique for studies of relevance to
fish osmoregulation, but also its potential redundancy for certain
experimental manipulations.
Chris N. Glover
185
Renal cell culture

The kidney of fish has two main roles in terms of osmotic and ionic
homeostasis. In freshwater, it forms a hypotonic urine as a means of
conserving ions and ridding the animal of osmotic water influx. In marine
environments, the kidney is primarily concerned with ion excretion and
water conservation. A number of cellular methods exist for examining the
function of renal cells from fish. Enriched membrane vesicle approaches
(e.g., Pane et al., 2006), and tubular perfusion techniques (see Beyenbach
and Dantzler, 1990) for example, have proven useful for elucidating
aspects of kidney function in fish. These preparations may have limited
applicability and utility for the study of piscine ionic and osmotic
homeostasis, depending on the aspect of osmoregulation under
consideration. Culture techniques also exist for renal proximal tubule cells
of the winter flounder (Pleuronectes americanus), a marine teleost.
Renal cell cultures of fish were developed primarily for the purpose of
investigating ion transport, and as such the utility of these preparations has
been well described (Dickman and Renfro, 1986; Renfro, 1995). This
method involves culturing on contractible collagen gels, which act to
enhance differentiation, while permitting experimental access to both
peritubular and luminal surfaces. The cells have established apical/
basolateral polarity and, when examined using Ussing chambers, are
observed to replicate transepithelial solute transfer in a manner similar to
that thought to occur in vivo (Dickman and Renfro, 1986; Dudas and
Renfro, 2001).
Recent investigations using this technique have yielded information
regarding the renal secretion of taurine, the important cell volume
regulator. Combining cell culture with electrophysiological methods
taurine secretion was found to be closely coupled to both extracellular
osmolarity and taurine levels (Benyajati and Renfro, 2000). Further recent
studies have investigated the roles of cortisol, Na+/H+ exchange and
carbonic anhydrase in renal sulfate excretion. By incorporating renal cell
culture with immunoblotting and molecular cloning, putative functions of
these entities in marine teleost proximal tubule cells were elucidated (Pelis
et al., 2003, 2005).
This technique has, therefore, proven to be a highly useful in vitro
model for examining renal function in marine teleosts. The diversity of
piscine kidney structure and function suggests the extension of this
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Fish Osmoregulation
preparation to other marine species, and, if applicable, to freshwater fish,

will provide new insights into renal function.
Intestinal cell/tissue culture
The intestine performs vital absorptive and excretory roles that act to
maintain osmotic balance. This is especially the case in marine fish where,
by virtue of the drinking response that compensates for diffusive ion loss,
the intestinal lumen is directly exposed to an ion-rich medium. A number
of in vitro techniques exist for examining the physiology of the piscine gut,
including intestinal perfusion, gut sacs, enriched membrane vesicles, and
isolated intestinal cells (see Glover et al., 2003a, b; Burke and Handy,
2005). Insofar as osmoregulatory investigations are concerned, these
methods may suffer from their lack of polarity, viability, and/or
homogeneity. Consequently, a piscine enterocyte cell culture model,
which overcomes many of these drawbacks, is of considerable utility.
There are literature reports of intestinal cell cultures from lamprey
(Ma and Collodi, 1996, 1999), but these remain almost completely
uncharacterized. Further attempts to culture intestinal cells from fish have
not yet yielded publishable results. Difficulties in intestinal cell culture are
not unique to fish, and may relate to the requirement of adequate feeder
layers to provide the necessary signals for cellular differentiation and
maintenance (Ali and Reynolds, 1996; Sambruy et al., 2001).
Recently, an intestinal tissue explant technique was reported for
sockeye salmon (Oncorhynchus nerka; Veillette and Young, 2005). These
authors demonstrated that explants from pyloric caecae and the posterior
intestine were responsive to cortisol, with effects on Na+/K+-ATPase
similar to those reported in other preparations at similar hormone levels.
However, the activity of Na+/K+-ATPase deteriorated over time, in
association with degradation of the basolateral membrane domain. This is
a novel method, and with further refinement and optimization of culture
conditions, it may offer considerable scope as a tool for investigating
osmoregulatory function in fish intestine.
There is significant heterogeneity between fish species, and indeed
along the gastrointestinal tract of fish, in terms of intestinal architecture
(see Ferraris and Ahearn, 1984). Species variation is primarily governed by
diet, and may be reflected in differences in epithelial folding and mucussecreting goblet cells for example. It remains possible that different fish
species, or explants from different sections of the digestive tract, may prove
Chris N. Glover
187
more amenable to culture. Ideally, an intestinal epithelial culture

technique will be developed in the future that allows culturing of a
lamellar epithelial surface to facilitate transport studies by permitting
manipulation of apical and/or serosal media in confluent cells with
polarity.
Skin cell/tissue culture
The role of the integument in salt and water balance is mostly overlooked
in favor of the relatively greater roles performed by the gills, gut and
kidney. The skin of fish can, however, play a significant role in addressing
the osmotic imbalance that exists between the animal and the external
milieu. This role extends from ion transport (e.g., Marshall et al., 1992;
McCormick et al., 1992) to the secretion of mucus that may act as a barrier
to diffusive ion and water movement across the body surface (Shephard,
1994). Whole skin preparations from frog have been essential in the
development of much of the current osmotic theory (e.g., Ussing and
Zerahn, 1951), and have been applied to great effect in fish (e.g., Marshall
et al., 1992). More recently, several cell culture techniques have been
developed for the examination of skin cell properties. These techniques
consist of the culture of skin cell explants (Mothersill et al., 1995), and a
primary skin cell culture (Lamche et al., 1998).
These preparations have been principally developed as
ecotoxicological tools, where the skinas a first line of defenceis a
toxicologically important barrier. Their success as in vitro tools may not,
therefore, necessarily depend upon the maintenance of functional
properties. As such, their value as osmoregulatory models is largely
undescribed. The explant technique is known to produce cells that
respond to cortisol (van der Salm et al., 2002). This indicates that
endocrine responsiveness remains intact, an important tenet of an in vitro
model. However, explants are unable to be cultured in lamellar sheets,
reducing their utility. Like early gill primary culture models, skin cell
cultures currently lack chloride cells, reducing their usefulness for
osmoregulation investigations. This may be overcome by applying doubleseeding techniques that have recently been established for chloride cell
incorporation in fish gills (see above).
To increase their utility, it may be possible to culture skin from
specialized skin regions. In the rainbow trout, for example, the skin on the
cleithrum bone appears to be relatively rich in chloride cells, and is likely
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Fish Osmoregulation
to perform a role in osmoregulation (Marshall et al., 1992). A further

example is the pectoral skin of mudskippers, which is reported as having
a high density of chloride cells that appear to function in chloride secretion
in seawater (Yokota et al., 1997). Difference in ion uptake between
chloride cells of the gill and skin are apparent (Flik and Verbost, 1993). It
is possible, therefore, that the greatest utility for skin cell culture
preparations lies in exploring these differences.
Primary cultures versus cell lines
In fish osmoregulation, cell lines based on piscine epithelia are less
commonly exploited than are primary cultures. They have, however, found
favor in fish toxicological and disease research (e.g., Castao et al., 2003;
Butler and Nowak, 2004). Cell lines are easier to maintain than primary
cultures, and thus offer benefits in terms of experimental and labor costs.
Additionally, a considerable advantage of cell lines is that they offer a
reproducible cellular phenotype. In contrast, freshly isolated cells are
prone to heterogeneity associated with cell culturing technique and
individual physiological condition (Castao et al., 2003). However, it is
generally considered that cell lines are less differentiated than primary
cultures, and thus may be poorly representative of the cells from which
they are derived.
In the few studies where piscine cell lines have been utilized to
investigate aspects of cellular ion and water transport, further
potential difficulties have emerged. In a comparative study, a rainbow
trout gill epithelial cell line was found to be less capable of generating
eicosanoids than either intact gill or freshly isolated cells (Holland et al.,
1999). Eicosanoids are important cellular signalling molecules with critical
roles in fish osmoregulation (Van Praag et al., 1987). Using a cyprinid
epithelial cell line (Epithelioma Papulosum Cyprini; EPC), standard culture
conditions were shown to result in vitamin E deficient cells (George et al.,
2000). This resulted in a reduced oxidative defence status in these cells,
and increased their susceptibility to toxicants. Deviations in other
metabolites, of critical importance to osmoregulatory function, may also be
induced by poor culture media. While media deficiencies are likely to be
deleterious to assessing physiologically relevant functions in both primary
culture and cell lines, the effects are likely to be more significant in cell
lines due to their extended culture histories. Further problems may also
exist in that raising medium osmolarity of the EPC cell line can result in
Chris N. Glover
189
enhanced apoptosis and cell death (Hashimoto et al., 1998). This indicates
that these cultures may be unsuitable for many of the manipulations that
may be of relevance to researchers of fish osmoregulation. This finding is
also in contrast to recent studies that show primary gill cell cultures
maintain physiological integrity in response to apical dilution (Zhou et al.,
2004), although their response to hypertonicity has not been investigated.
Studies that depend more stringently on physiological parameters that
resemble those in intact organisms and tissues are likely to be better served
by primary cultures. However the genetic homogeneity of cell lines is likely
to offer some advantages over primary cultures in terms of molecular
endpoints. The EPC cell line has, for example, been successfully used to
isolate a taurine transporter that is likely to perform a role in cell volume
regulation (Takeuchi et al., 2000).
Purifying Epithelial Cell TypesTechniques for Assigning
Osmoregulatory Function
Considerable difficulties exist with the assignation of physiological
function to specific cell types within multifunctional epithelia of mixed cell
populations. Consequently, in the fish gill, several controversies pertaining
to ion transport exist. With regard to sodium transport, for example, both
the nature of the transporters involved (see Cloning of transporters;
Fig. 7.1) and the roles of pavement and chloride cells in this process, are
unresolved. To address these issues, methods are required that permit the
examination of individual cell types. Based on cell isolation methods in
mammalian epithelia, techniques have recently been developed in fish
that are capable of isolating cell types from the gill. Following isolation
these cells remain viable for molecular, biochemical and physiological
investigation. These tools have been integral to studies that have ascribed
biological function to cell type, and to research that has challenged
traditional branchial cell type classification.
The experimental utility of relatively pure chloride cell populations
has been recognized for some time. With this goal in mind, a number of
different isolation techniques have been used (e.g., Sargent et al., 1975;
Naon and Mayer-Gostan, 1983; Perry and Walsh, 1989). These methods
suffered from either one or a combination of difficulties relating to low
purity, reduced viability, and poor applicability to both freshwater and
marine fish. Recently, a Percoll density gradient centrifugation technique
190
Fish Osmoregulation
Fig. 7.1 Schematic diagram illustrating how molecular and cellular advances have altered
our understanding of sodium transport in freshwater rainbow trout. Pharmacological
(A) and immunological (B) studies may produce conflicting results, and have often been
unable to attribute a transport function to a particular cell type (see text for discussion). The
models demonstrated here are primarily based on data from Lin and Randall (1993) and
Wilson et al. (2000a). The development of cell isolation protocols and molecular cloning of
transport proteins has considerably altered these models (C). This hypothesis of sodium
transport awaits further molecular and immunolocalization confirmation. See Molecular
cloning of transporters, Purifying epithelial cell types and Pharmacology of ion transport
for details.
was developed that resulted in highly pure (89-98%) populations of

pavement and chloride cells from eel gill (Wong and Chan, 1999b). The
low cytotoxicity and adjustable osmolarity of Percoll as a substrate resulted
in viable cells that displayed biochemical and morphological properties
resembling those in vivo (Wong and Chan, 1999b). This technique has
been applied with success to eel and rainbow trout from both freshwater
and marine environments (Wong and Chan, 1999b; Goss et al., 2001;
Hawkings et al., 2004).
Chris N. Glover
191
A significant finding from these initial studies of isolated gill cells was
the elucidation of two mitochondria-rich cell types (Wong and Chan,
1999b; Goss et al., 2001), confirming earlier morphological observations
(e.g., Pisam and Rambourg, 1991). The nature, and scientific implication,
of mitochondria-rich subtypes will be discussed below. Methodologically,
the presence of two mitochondria-rich cells required an additional
isolation step. In eel, gill cell subtypes were further separated by flow
cytometric techniques. Variation in cell properties such as size, granularity,
and the autofluorescence of mitochondria-associated metabolites,
permitted these cells to be adequately sorted into homogenous
populations. Furthermore, changes in these properties were used to
monitor the alterations that occurred in these cell populations as the
animal were acclimated to seawater or exposed to cortisol (Wong and
Chan, 1999a, 2001). Laser scanning cytometry, a related but more
sophisticated technique, has also been applied to separate and assess the
effects of salinity changes on specific cell types of the killifish gill (Lima and
Kltz, 2004).
In rainbow trout, flow cytometry was not a viable method of isolating
these cell types (Galvez et al., 2002), suggesting that species differences are
likely to exist in cellular properties amenable to flow cytometry. Instead, it
was found that the two trout mitochondria-rich cell subtypes could be
distinguished on the basis of their affinity for peanut lectin agglutinin
(PNA; Goss et al., 2001). One population of mitochondria-rich cells
bound PNA, the other did not. The mitochondria-poor cells (pavement
cells) already separated by centrifugation, also failed to exhibit PNA
binding. PNA specifically binds to glycosylated membrane proteins with a
terminal D-galactosyl residue, and this property has previously been used
to distinguish between intercalated cell subtypes in mammalian kidney
(LeHir, 1982). Fluorescein isothiocyanate (FITC) conjugated-PNA, and
anti-FITC antibodies conjugated to iron particles were then used in
combination with magnetic separation techniques to isolate the two
mitochondria-rich cell subtypes in trout (Galvez et al., 2002). Cells were
initially incubated in PNA, then in the antibody, before being passed
through a magnetic field. Those cells without PNA affinity (nonmagnetic) were flushed through, while PNA-positive cells (with iron
particle) were trapped, and later eluted. This technique resulted in viable,
homogenous cell populations that can be utilized for subsequent analyses.
This isolation protocol has been applied, for example, to investigate the
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Fish Osmoregulation
relative changes in distinct cell population enzyme activities that occur

with seawater acclimation (Hawkings et al., 2004).
From a functional perspective, gill cells have been traditionally
considered as either pavement or chloride cells. Categorization is usually
made by virtue of simple bioassays based on the higher mitochondrial
content of the latter cell type. The presence of two mitochondria-rich cell
types, with properties distinct enough to permit their successful
separation, suggests that the situation is more complicated. Supported by
mitochondrial density data and the nature of cellular changes associated
with seawater exposure, Wong and Chan (1999a) concluded that the
additional mitochondria-rich cell type in eel was a chloride cell.
Contrastingly, based on transmission electron microscopy and differential
PNA binding, this additional subtype was classified as a mitochondria-rich
pavement cell (Goss et al., 2001). A number of possibilities for such a
discrepancy exist. Some assays used for characterizing cell types are more
robust than others and this could have influenced the findings.
Alternatively, the different species used may account for these differences.
The possibility of undiscovered mitochondria-rich pavement cells in eel,
and chloride cells in trout, would also influence the conclusions drawn.
Irrespective, it is clear that cell subtypes exist in fish gill.
Isolation of cell subtypes has significant implications for many existing
controversies in fish osmoregulation. Of particular relevance is the
reassessment of branchial sodium transport in freshwater fish (see Fig. 7.1).
One theory states that sodium uptake is achieved via an epithelial sodium
channel (ENaC), and driven by an apical proton pump (H+-ATPase; see
also Cloning of transporters). Immunological evidence has demonstrated
the presence of a V-type H+-ATPase either exclusively in pavement cells
(Sullivan et al., 1995), or in both pavement and chloride cells (Wilson
et al., 2000a, b). Galvez and colleagues (2002) attempted to address this
incongruity using their isolated cell technique in rainbow trout. Following
cell separation, mitochondria-rich cells that were PNA negative (i.e.,
mitochondrial-rich pavement cells) exhibited protein expression levels of
H+-ATPase that were two-fold greater than those of chloride cells. These
mitochondria-rich pavement cells also demonstrated a relative increase in
proton pump expression under conditions that promote acid excretion
(hypercapnia). In an additional study, cell isolation was used in
combination with pharmacological evidence to suggest the presence of an
ENaC in the same cell type (Reid et al., 2003). These results indicate that
Chris N. Glover
193
the mitochondria-rich pavement cell may be the branchial location for

acid-mediated sodium uptake. It is interesting to note that in the
mammalian kidney, of the two mitochondria-rich cell subtypes, the one
that is PNA-negative is also thought responsible for acid secretion,
analogous to the scenario in trout gill (Reid et al., 2003). This finding
appears to resolve the seemingly conflicting immunological evidence. If
the mitochondria-rich pavement cell is the primary locus for branchial
sodium uptake, then under more simplistic categorization it could be
classified as either a pavement cell (based on morphology) or a chloride
cell (based on mitochondrial content), leading to either of these cell types
being attributed with sodium transport properties.
Cell isolation protocols do have some significant drawbacks. Isolated
cells tend to lose their apical/basolateral polarity (Galvez et al., 2002; Reid
et al., 2003). This may result in the redistribution of ion transport
structures and functions, and will limit the usefulness of these techniques.
Furthermore, the passage of ions across epithelia is often dependent upon
cooperation between epithelial cell types (Do and Civan, 2004). As such,
transport may not function in isolated cells, as it does in epithelia.
Nevertheless, such methods offer considerable promise, especially in
conjunction with other molecular and cellular techniques. The custom
seeding of primary gill cell cultures with specific isolated cell types, for
example, will overcome polarity problems, and could be a tool of some
significance for examining transport function. In addition, this preparation
has great potential for molecular cloning studies, where entities involved
in ion transport could be assigned to particular cell populations. Advances
in RNA isolation and amplification technology (Ginsberg, 2005), are likely
to overcome many of the technical difficulties associated with such
methods.
Enriched Membrane VesiclesMechanistic and
Structural Analysis of Transport
The ability to preferentially enrich and encapsulate a specific membrane
component of an epithelium is of considerable value. It enables both the
localization of specific functions to a membrane surface, and the
mechanistic characterization of epithelial processes (Berteloot and
Semenza, 1990). Enriched membrane vesicles permit the delineation of
osmoregulatory processes in a system relatively free from a number of
confounding impacts. These include factors such as the unstirred water
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Fish Osmoregulation
layer of epithelial surfaces, transport systems on other membranes, and

systemic feedback. While these are physiologically important features of
epithelia, they can preclude measurement of transport mechanism.
Membrane vesicles are ideally suited for kinetic analyses. They also offer
the advantage of being able to manipulate intracellular and extracellular
constitution, in terms of both ion composition and the application of
pharmacological agents. Of particular benefit is the capacity to examine
function in the native membrane.
These advantages have been extensively capitalized upon in studies of
both mammalian and piscine epithelia. Enriched membrane techniques
have been applied to gill, kidney and intestine of fish, with a particular
focus the characterization of divalent ion transport (Bijvelds et al., 1996;
Flik et al., 1996). In contrast, examination of the strong osmoregulatory
ions, sodium, potassium and chloride across isolated membrane
preparations, has been comparatively neglected (although see De Giorgi
et al., 1992).
Methodological considerations are likely to explain this lack of
research activity. The osmotic reactivity of these sealed vesicle
preparations, for example, may be mitigating. The investigation of
transport processes may require manipulation of electrochemical
gradients, which can result in altered intravesicular space as the vesicles
shrink or expand. Furthermore, radiotracers or ion-specific dyes used to
monitor ion movement rapidly equilibrate owing to the small
intravesicular volume. Consequently, it may be difficult to monitor the
initial rate of ion transfer, a measurement that is favored owing to its
independence from properties such as vesicle size (Berteloot and Semenza,
1990). However, these problems are not significant and can often be
overcome by experimental advances such as stop-flow fluorimetry, which
permits assessment of transport over fractions of a second (e.g., Rivers
et al., 1998). In other comparative models, membrane vesicles have offered
key insights into strong ion transport (e.g., Towle, 1993; Dubinsky et al.,
2000). These preparations are, therefore, still likely to be useful for the
investigation of epithelial ion movement in fish. It is important to note
that the ability to isolate enriched and sealed vesicles of suitable purity
appears to be technique- and investigator-dependent, and that certain
membranes and epithelia are more suited to this application than others.
Apical (brush-border) enrichment of the intestine and basolateral
enrichment of the gill, for example, appear to be the most robust
preparations.
Chris N. Glover
195
Enriched membrane studies may also have significant scope for the
investigation of membrane lipids and their roles in osmoregulation.
Basolateral membrane isolation techniques have been used to investigate
the role of specific membrane phospholipid microdomains on the activity
of vital osmoregulatory enzymes in fish gill (Lingwood et al., 2004, 2005).
Findings from these studies suggest that migration of Na+/K+-ATPase to
sulfogalactosylceramide-rich microdomains is responsible for the
upregulation of this enzyme during acclimation to seawater (Lingwood
et al., 2005). The importance of lipids in osmoregulation has been
suggested by studies that show lipid composition changes with salinity
acclimation (Hansen et al., 1999; Hansen and Grosell, 2004), but until the
utilization of enriched membrane techniques the significance of this was
not well understood.
Membrane isolation also facilitated the discovery of the unusually high
cholesterol to phospholipid ratio in the basolateral membrane of a marine
elasmobranch gill (Fines et al., 2001). This finding may explain the low
urea permeability of the elasmobranch gill, which is partially responsible
for the ability of these animals to maintain this important osmolyte against
a strong diffusive gradient for urea loss. Recent investigations, utilizing
membrane vesicles from both teleost and elasmobranch gill (Hill et al.,
2004), and from shark rectal gland (Zeidel et al., 2005), are somewhat
contradictory. These studies suggest that the low permeability to urea and
water it is not due to lipid composition. Instead, it is proposed that factors
such as vascular perfusion, unstirred water layers (Hill et al., 2004), or
specific protein components (Zeidel et al., 2005) may play an essential role.
Studies have also demonstrated the presence of specific urea transporters
in the branchial basolateral membrane vesicles of both sharks and teleosts
(Fines et al., 2001; Walsh et al., 2001; McDonald and Wood, 2004). These
transporters would act to pump urea from the gill back into the circulation,
thus reducing apparent urea permeability. Irrespective of the factors
involved, the efficacy of membrane vesicles in the investigation of urea
transport is clear.
The examples described above illustrate the utility of membrane
vesicle preparations for the analysis of aspects of salt and water balance in
fish. As with most techniques, however, there are important experimental
caveats that need to be overcome. The purity of the vesicle population, the
orientation of the membrane (right-side-out versus inside-out), and
confirmation of osmotically active vesicles are important details, that are
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Fish Osmoregulation
all too frequently unreported. Appropriate marker enzymes can be used to

confirm purity and orientation. However, regional heterogeneity of
enzyme activity and content (Almansa et al., 2001; Dopido et al., 2004)
may influence the conclusions drawn. For this reason, it may be necessary
to carefully characterize the marker enzymes of choice. A recent study
labelled the apical surface of winter flounder gills with a biotin derivative
(Senz et al., 2003). After employing the membrane isolation protocol, the
putative apical membrane fraction was incubated with avidin, and biotin
content was determined via the peroxidase assay. The simultaneous
measurement of the proposed apical membrane marker ADPase correlated
well with the biotin-avidin results. This established the suitability of using
ADPase activity as a marker of branchial apical membranes in this species.
With the expanding array of molecular and cellular techniques, the
use of enriched membrane vesicles is perhaps an overlooked method. Data
from several recent studies indicate their utility. Membrane vesicle
preparations may offer an important bridge between the structural
information resulting from molecular cloning of transporters, and their
physiological roles in osmoregulating epithelia.
Pharmacology of Ion TransportCaveats for
Comparative Biologists
There are hundreds of pharmacological agents that can be used to block
aspects of ion and water transport. These range from natural toxins to
synthetic chemicals, with actions varying from those on second messenger
systems to those affecting the transporters themselves. Many of these
agents are targeted as therapeutics for human disease, and although a
widely utilized and valuable tool for elucidating transport mechanisms in
comparative physiology, their applicability to such models is rarely
questioned.
The mechanism of sodium transport across the fish gill is an ongoing
controversy. Based partly on pharmacological evidence the favored model,
at least during the 1990s, stated that uptake was achieved through an
epithelial sodium channel (ENaC), powered by an apical proton pump (Vtype H+-ATPase; Avella and Bornacin, 1989; Lin and Randall, 1991,
1993; see also Cloning of transporters below; Fig. 7.1). More recently,
other evidence has supported the alternative hypothesis, that of an apical
sodium-proton exchanger (NHE). The ongoing controversy perhaps
warrants a critical re-examination of the foundation for pharmacological
transporter blockade.
Chris N. Glover
197
Amiloride is a widely used pharmacological agent that inhibits the

function of the NHE. Some NHE isoforms, including the major apical
branchial NHE found in fish (NHE3; Choe et al., 2002, 2005; Hirata et al.,
2003), remain relatively resistant to blockade (Masareel et al., 2003).
Amiloride is somewhat promiscuous, and will also block both ENaC and
the sodium/calcium exchanger (Masareel et al., 2003), and may disrupt
cellular signalling pathways (Holland et al., 1983). Furthermore, amiloride
is known to promote the binding of atrial natriuretic peptide to its receptor
(De Lean, 1986). This effect could modify sodium and water transport
independently of direct blocking effects on transport moieties in systemic
preparations. As a result, the interpretation of amiloride actions cannot
necessarily be restricted to consideration of its impact on NHE.
There are still important roles for pharmacological agents in the study
of fish osmoregulation. To a large degree, the promiscuity of amiloride can
be minimized by the careful choice of dose. Furthermore, the development
of more specific inhibitors (e.g., phenamil, HOE-694) has eliminated many
of the known non-specific actions. Despite these measures, precautions
must be taken to ensure the suitability of pharmacological studies. A major
challenge is the heterogeneity of the biological material available for
osmoregulatory investigation. This can display significant variation in
properties depending on fish species, osmoregulatory tissue, and the
physiological state of the source animal. In addition, it may be simplistic
to expect that pharmacological agents applied to mammalian preparations
will have identical actions in fish. It is known, for example, that some fish
NHEs have unique phenotypes (Nickerson et al., 2003). This may lend to
them properties that alter the efficacy of pharmacological attack.
Subsequently, considerable investigation of inhibitor activity is required
prior to use, a step rarely undertaken (cf., Reid et al., 2003).
Amiloride is one of the better characterized pharmacological agents
used in fish studies. It may be that its wide array of non-specific cellular
actions is a case of familiarity breeds contempt. The more that is known
about a blocker, the more likely it is that promiscuous actions can be
ascribed. Thus the new generation of pharmacological agents, while
appearing to exhibit fewer non-specific actions, may have effects on nontarget pathways that remain undiscovered. It is also worthwhile
highlighting the fact that variations in isoforms may also continue to
influence effects of these new blocking agents. HOE-694, for example, is
touted as a useful agent against NHE, and has a high specificity for this
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Fish Osmoregulation
transporter. However, it is only a poor inhibitor of NHE3 (Masareel et al.,

2003), the main branchial isoform in fish (Choe et al., 2002, 2005; Hirata
et al., 2003).
Ouabain, a plant alkaloid, is another commonly exploited
pharmacological tool in fish osmoregulation. It is a potent inhibitor of the
basolateral Na+/K+-ATPase that is responsible for driving transepithelial
ion transport. Following the discovery of ouabain as a circulating hormone
in mammals (e.g., Mathews et al., 1991), endogenous immunoreactive
ouabain was subsequently identified in fish plasma, where its levels
fluctuate in response to salinity changes (Kajimura et al., 2004). The
primary cellular target of ouabain appears to be Na+/K+-ATPase.
However, effects of this hormone on the release of the important
osmoregulatory hormone prolactin have been observed at levels less than
those required for effects on the sodium pump (Kajimura et al., 2005).
Ouabain may also have direct impacts on cellular signalling pathways, at
least in mammals (Harwood and Yaqoob, 2005). This suggests that
ouabain could exert biologically relevant impacts on endogenous
pathways, beyond those previously attributed to effects arising from
inhibition of Na+/K+-ATPase. This may have significant consequences for
the use of ouabain in fish osmoregulation, and again suggests that results
arising from the use of pharmacological agents in comparative models have
to be carefully interpreted.
MOLECULAR BIOLOGY OF FISH OSMOTIC AND IONIC
REGULATION
Fish are the largest, and arguably the most successful, extant group of
vertebrates. The diversity of fish species, and the environments they
inhabit, provides an ideal opportunity for examining the mechanisms of
ionic and osmotic homeostasis. In particular, it permits assessment of how
alterations in the osmoregulatory apparatus have contributed to this vast
radiation. Their ability to thrive in highly saline, extremely dilute, acidrich or strongly alkaline waters depends, to a large extent, on the
adaptability of the osmoregulatory apparatus. The genetic material
encoding the ion transporters, hormones, endocrine receptors and signal
transducers can differ considerably between even closely related fish
species. Differences in the number, the nature, and the regulation of these
moieties will have significant impact upon the processes that enable salt
Chris N. Glover
199
and water balance, and may explain how fish have been able to adapt to
such a wide range of osmotic environments.
Our knowledge of the differences in genomes, genes, proteins and
metabolites between fish species is poor. Our understanding of how these
differences may influence fish osmoregulation is even less well developed.
This ignorance is due in part to a lack of bioinformatics information
related to fish. Progress is being made on a selected few species (see Table
7.1), with detailed sequence knowledge of the zebrafish and pufferfish
genomes representing a significant advance. However, the phylogenetic
distance between these species and those that are often considered of
greater relevance to the fish physiologist is often considerable.
Theoretically, the tremendous diversity in form, habitat, physiology and
genetics of fish, is of great benefit for examination of osmoregulatory
structures and processes. Practically, however, it may represent an
important obstacle to the elucidation of molecular processes.
Despite the difficulties associated with utilizing molecular tools in fish
osmoregulation, considerable progress has been made. The use of cloning
techniques has provided structural support for the ion-transporting
theories established from physiological data. It has also offered insight into
Table 7.1 Genetic resources for the piscine molecular biologist.
Sequencing projects
Website(s)
Zebrafish (Danio rerio)

Japanese pufferfish (Fugu)
http://www.sanger.ac.uk/Projects/D_rerio/
http://genome.jgi-psf.org/Takru4/Takru4.home.html
http://fugu-sg.org/
http://www.genoscope.cns.fr/externe/tetranew/
http://dolphin.lab.nig.ac.jp/medaka/
http://salmongenome.no
http://web.uvic.ca/cbr/grasp
http://www.genome.gov/12512292
Green-spotted pufferfish (Tetraodon)

Japanese medaka (Oryzias latipes)
Atlantic salmon (Salmo salar)
Threespine stickleback
(Gasterosteus aculeatus)
Rainbow trout
(Oncorhynchus mykiss)
http://dga.jouy.inra.fr/cgi-bin/lgbc/main/.pl?base=rainbow
Other resources
Genbank (Nucleotide database
for all taxa)
Swiss-Prot (Proteomic database
for all taxa)
Zebrafish gene resources and links
Medaka resources and links
http://www.ncbi.nlm.nih.gov
http://www.expasy.ch/sprot
http://zfin.org
http://biol1.bio.nagoya-u.ac.jp:8000/
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Fish Osmoregulation
unforeseen complexities in endocrine regulation of fish salt and water

balance. Examples of these advances will be discussed below. The potential
use of methods for manipulating and monitoring cellular expression will
also be addressed. The utilization of molecular biology in fish
osmoregulation has significant pitfalls, but the opportunities it offers in
terms of new discoveries are unrivalled.
Physiological versus Genomic ModelsAs the Krogh
Flies
The choice of fish species in studies of piscine salt and water balance has
been driven extensively by Kroghs principle. This suggests that for any
biological question, nature provides an experimental system best suited for
its investigation (Krebs, 1975). Osmoregulation research has generally
focussed on those animals with the ability to acclimate to altered
environmental osmolarity, or which undergo life history- or migrationrelated changes exposing them to vastly different ionic and osmotic
challenges. The changes in epithelial structure and physiology that
accompany this acclimation have been invaluable for our understanding of
the mechanisms that are responsible for, and which regulate, osmotic and
ionic homeostasis (Wheatly and Gao, 2004). More practical concerns
ensure those species that are easily cultured and of economic importance
are especially favored.
The application of Kroghs principle in fish genetics has seen the
development of model organisms such as the Japanese pufferfish (Takifugu
rubripes; Fugu), a fish with a compact genome densely populated with
coding sequences (Brenner et al., 1993). The relatively small genome size
of Fugu has made it a target for full genome sequencing (Aparicio et al.,
2002). These attributes facilitate studies examining a wide range of
biological phenomena, including the importance of non-coding DNA
(Woolfe et al., 2005), gene regulation (Goode et al., 2005), and even
investigation of human genetic disease (Yu et al., 2005). By virtue of the
genetic resources available, this fish is a model species of considerable
importance. However, this species is not readily available for laboratory
culture in many areas of the world. Its stenohaline nature also renders it
inappropriate for many of the investigations of greatest interest to fish
osmoregulation. The genome sequence of the zebrafish is also near
completion, an effort driven mainly by the value of this fish as a
developmental biology model. While this is a more accessible species, it
too lacks applicability for investigating salt and water balance.
Chris N. Glover
201
There are, however, increasing amounts of gene information available

for other fish species, in the form of expressed sequence tag and cDNA
sequencing projects (see Table 7.1). Consequently, with time, the available
sequence information for physiological Kroghs models will increase. This
will significantly increase the applicability of molecular techniques to
investigators of fish osmoregulation. Even the genomes that have already
been sequenced are of some value. These can provide, in some cases, an
excellent starting point for homology-based cloning or transcriptomic
studies, and for bioinformatic database mining approaches (see below, also
Wheatly and Gao, 2004).
It has been suggested that the Japanese medaka (Oryzias latipes) may
offer greater utility as both a physiological and genetic model (Inoue and
Takei, 2003). This is a well-established transgenic model, and considerable
sequence information is available (Tanaka and Kinoshita, 2001; Inoue and
Takei, 2003). Essentially a freshwater species, O. latipes can be acclimated
to different salinities. It will, for example, spawn, hatch and undergo
embryonic development in seawater. However, adult survival is relatively
poor, and is only achieved by careful acclimation (Inoue and Takei, 2002).
This suggests changes that may be interpreted as osmotic acclimation may
better represent generalized stress effects. Of greater applicability is the
presence of closely related species that naturally occur in varying salinities
(Inoue and Takei, 2003). Comparison of molecular responses and
expression between species with closely related phylogenies may yield
insights into the mechanisms and fundamental processes linked to the
ability to adapt to different salinities (Inoue and Takei, 2003).
Currently, a distinction exists between the organisms of greatest
interest to the molecular biologist and those with greatest applicability to
the physiologist. This is a disparity that is rapidly diminishing as sequence
information in species with greater utility for the examination of fish
osmoregulation increases. Future sequencing efforts will be further aided
by the continuing advances in tools that improve speed, cost and accuracy
of genome sequencing (Bonetta, 2006). Even with the existing datasets,
the scope for molecular investigations of fish osmoregulation has increased
significantly over the previous decade. In another decade from now, many
of the arguments regarding the lack of genetically characterized models of
relevance to piscine salt and water balance will very likely be redundant
such is the rapid advance of this field.
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Fish Osmoregulation
Gene Duplication and Isoform SwitchingThe

Unravelling Complexity of Fish Biology
Gene duplication represents a significant challenge for the piscine
molecular biologist. There is considerable support for a whole genome
duplication event that occurred early in the teleost lineage (Christoffels
et al., 2004; Jaillon et al., 2004). The resulting multiple gene copies are
believed to be partially responsible for an enhanced adaptive capacity and,
consequently, may have promoted the radiation of teleost fish (Venkatesh,
2003). The genome of fish remains relatively plastic, and evidence of
localized duplication events effecting osmoregulatory genes exists (e.g.,
Rajarao et al., 2001; Inoue et al., 2003). These localized gene duplications
may be restricted to species or certain lineages of teleosts. Additionally,
some fish have lost gene copies arising from duplication events, further
contributing to the molecular complexity of osmoregulation in fish. The
result of this genetic flux is a gene- and species-specific distribution of gene
duplicates.
A number of osmoregulatory moieties with gene duplicates have been
identified in fish. The V-type H+-ATPase B subunit (Niederstatter and
Pelster, 2000) and an aquaporin isoform 1 (Cutler and Cramb, 2001) in eel
are represented by duplicated coding sequences. The multiple copies of
Na+/K+-ATPase = and > subunit isoforms in zebrafish (Rajarao et al.,
2001), rainbow trout (Richards et al., 2003) and Atlantic salmon (Gharbi
et al., 2005) are likely to have arisen by gene duplication. The complicated
natriuretic peptide system in medaka and pufferfish is proposed to have
resulted from both whole genome duplication and local tandem
duplication events (Inoue et al., 2003).
The physiological implications of possessing an assortment of closely
related genes are many and varied. The presence of duplicate coding
sequences may spawn new functions for an otherwise superfluous gene.
The duplicate copies of the midkine growth factor genes in zebrafish, for
example, have different functional properties than their mammalian
counterpart (Winkler et al., 2003). Additionally, fish antifreeze proteins
are believed to be derived from a gene duplicate originally coding for the
protease precursor trypsinogen (Chen et al., 1997).
There is also evidence that gene duplicates of ion transport proteins
can be independently regulated. Richards and colleagues (2003) identified
three Na+/K+-ATPase =1 subunit copies in rainbow trout, denoted as
=1a, =1b and =1c. The tissue distribution of =1a and =1b was similar,
Chris N. Glover
203
indicating a conserved functional role. Upon exposure of trout to seawater,

these two transcripts were differentially regulated. While =1a expression
decreased in the gill, =1b expression increased (Richards et al., 2003). This
isoform switching is likely to be controlled hormonally, as it accompanied
the cortisol surge observed after seawater transfer (Richards et al., 2003).
The opposite pattern (an increase in =1a expression) was observed in
sockeye salmon that migrated from seawater to freshwater (Shrimpton
et al., 2005). These studies indicate the presence of differential
osmoregulatory gene duplicate regulation and also suggest that subtle
differences between these duplicated genes could offer functional
advantages, depending on osmotic environment.
In mammals, four distinct =, and three > subunits exist, that can
combine in order to generate a functional sodium pump (Blanco, 2005).
Orthologs for some, but not all, of these subunit isoforms have been
discovered in fish (see Gharbi et al., 2005). The functional Na+/K+ATPase is a heterodimer, consisting of a single = and a single > subunit,
with other regulatory elements playing an important role (e.g., Blanco,
2005). The variety of subunit isoforms and the gene duplication of a
number of these isoforms indicates the potential for a vast assortment of
different heterodimers in fish. These are likely to be expressed in a celland tissue-dependent manner. The possibility that each heterodimer has
distinct functional and regulatory properties (see Richards et al., 2003;
Deane and Woo, 2005), hints at the previously unrealized complexity of
this protein in fish osmoregulatory epithelia.
Independent regulation and isoform switching of Na+/K+-ATPase has
important consequences for the monitoring of this enzyme in
osmoregulation studies. Commonly used assays may greatly oversimplify or
even misrepresent experimental changes in this protein (Richards et al.,
2003). For example, the measurement of Na+/K+-ATPase activity in a
tissue homogenate may represent the summed activities of a number of
different heterodimers. The overall effect may, therefore, mask the impact
on individual heterodimers. Gene expression assays are, thus, likely to offer
a more sensitive measure of cellular Na+/K+-ATPase dynamics (e.g., Scott
et al., 2004).
Na+/K+-ATPase is a key enzyme in fish osmoregulation, but
represents only a single point in a single pathway influencing ion and water
movement across osmoregulatory epithelia in fish. It is possible that other
components of ion transport pathways are represented by differentially
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Fish Osmoregulation
regulated duplicate genes and also exhibit isoform switching. Given all the
elements that may combine to ensure ionic homeostasis, the complexity of
these processes is likely to be extensive.
Cloning of TransportersMolecular Support for
Physiological Theories
Physiological studies can provide only circumstantial evidence for the
existence of specific transporters and ion uptake pathways. Recent efforts
have utilized molecular cloning techniques to establish a structural basis
for ion transport in fish epithelia. Over the past decade the presence of ion
channels (e.g., cystic fibrosis transmembrane conductance regulator),
bicarbonate transporters, sodium/proton exchangers, chloride
cotransporters, proton pumps (V-type H+-ATPase), sodium pumps (Na+/
K+-ATPase) and water channels (aquaporins), has been elucidated (see
Cutler and Cramb, 2001; Hirose et al., 2003; Perry et al., 2003 for reviews).
Structural evidence has permitted reassessment of many of the ion
transport hypotheses established by previous physiological investigations.
For example, evidence regarding the nature of proton-facilitated sodium
transport in the freshwater fish gill is contradictory. Uncertainty extends
to both the likely cellular location of this transport process (see Purifying
epithelial cell types above), and the structural entities involved (see
Kirschner, 2004). Two physiological models have been proposed. In one,
sodium exchange for a proton is mediated by an apical sodium/proton
exchanger (NHE). This model relies on the creation of a favorable
electrochemical gradient for sodium influx, likely generated by the actions
of the basolateral Na+/K+-ATPase, and the efflux of protons down a
concentration gradient. Calculations based on the few existing measures
of intracellular sodium and pH suggest this model may not viable, at least
in rainbow trout (Avella and Bornancin, 1989). An alternative model
states that an apical proton pump (H+-ATPase), in concert with the
basolateral Na+/K+-ATPase, generates an electrochemical gradient that
favors the inward passage of sodium via an epithelial sodium channel
(ENaC). This model is supported on the basis of physiological and
pharmacological evidence (Lin and Randall, 1991, 1993).
Immunological studies aimed at investigating ion transporters in gill
epithelia provided ambiguous data. Supporting the favored model was
evidence demonstrating the presence of proton pumps on apical branchial
surfaces, and sodium pumps on the basolateral surface of branchial
Chris N. Glover
205
epithelial cells (e.g., Wilson et al., 2000a, b). In some species, however,
these activities were not located to the same cell types (Piermarini and
Evans, 2001; Tresguerres et al., 2005), contradicting the model.
Conversely, in tilapia and rainbow trout, a putative ENaC was discovered
in the same cell type as the proton pump (Wilson et al., 2000a), favoring
the pump/channel coupling hypothesis. The presence of an apical NHE
has been discerned in some species (Wilson et al., 2000b; Edwards et al.,
2002; Choe et al., 2005). While this does not necessarily contradict the
model, it does suggest the alternative hypothesis involving an NHE should
bear further consideration. In general, the findings from immunological
studies were somewhat confusing, and suggested that transport
mechanism could be species dependent.
Pharmacological and immunological evidence is subject to several
pitfalls. Some of these have been detailed above (see Pharmacology of ion
transport). In immunological studies, antibodies raised to different
subunits or regions of the protein of interest can give different results. For
instance, the presence of the ENaC >-subunit, but not the =-subunit, can
be detected in fish gill (Wilson et al., 2000a). Furthermore, the use of
heterologous antibodies may cause misleading conclusions. Katoh and
colleagues (2003), for example, showed a basolateral locus for the proton
pump using a homologous antibody in killifish, in disagreement with apical
locations from studies utilizing heterologous antibodies (e.g., Wilson et al.,
2000a, b). Molecular cloning of transport proteins provides a structural
framework in which to interpret the results of these studies.
Molecular cloning complements physiological, pharmacological and
immunological approaches, and can be used as a tool to explain some of
the apparent experimental contradictions that may arise from these other
techniques. A vacuolar-type H+-ATPase has been cloned from the gill of
a number of fish species (Perry et al., 2000; Boesch et al., 2003; Hirata et al.,
2003; Katoh et al., 2003), lending support to the physiological,
pharmacological, and immunological evidence for its existence in this
tissue. Likewise, a number of sequences now exist for NHE isoforms in fish
(e.g., Choe et al., 2002, 2005; Hirata et al., 2003). Of particular interest is
the NHE3 isoform which appears to be the major apical form in fish gills
(Choe et al., 2002, 2005; Hirata et al., 2003). Comparisons between the
stingray and zebrafish NHE3 show only a 53% amino acid identity between
the predicted protein sequences of these two species. Such divergence may
explain the core difficulty with the use of non-homologous antibodies
(non-homologous antibody recognition sites).
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Fish Osmoregulation
The fact that sequences exist in fish species for components of both of
the competing models in freshwater fish gill provides no clue as to which
is most likely to explain sodium transport. Of more interest is the
evidential failure to clone a piscine ENaC. No sequences from the
completed pufferfish genome share homology with cloned ENaCs from
mammalian species. Even though pharmacological and immunological
evidence has suggested the presence of ENaC in fish gill, it is difficult to
support the existence of a proton pump/sodium channel coupled model
without the structural basis for these moieties in the genome of fish. It is
possible that attempts to clone ENaC may be successful in other species,
but there is no molecular support for its occurrence in any fish examined
to date.
Cloning studies offer important supporting evidence for the presence
of ion transporters in osmoregulating epithelia. As such they provide the
basis for investigations exploring both the cellular localization and
physiological roles of the cloned gene products. Integration of cloning
studies with other molecular and cellular methods is, therefore, likely to
significantly enhance our existing knowledge of fish osmoregulation.
Corticosteroid Hormone ReceptorsMolecular Insight
into Endocrine Regulation
The hormonal control of salt and water balance in fish is complex. A wide
variety of endocrine, paracrine and autocrine factors have been identified
over recent years which can act as effector molecules on various aspects
of osmotic regulation. Using homology-based cloning, many hormones,
their synthesizing enzymes, and their receptors have now been sequenced
in fish, and homologous assays have been established to accurately
determine the circulating levels of endocrine factors in fish blood and
tissues. These molecular investigations have often yielded new
information regarding the ancestral role of these hormones, and also
provided insight into aspects of ion regulation in other vertebrate systems.
It is not the scope of the current chapter to summarize these findings as
this has been the focus of a number of recent reviews (e.g., Mommsen et
al., 1999; McCormick, 2001; Sakamoto et al., 2001; Evans, 2002; Manzon,
2002; Takei and Hirose, 2002). This section will, instead, concentrate on
the intracellular steroid receptors of the corticoid hormones. In this area,
recent advances in molecular techniques have produced surprising
findings that highlight the complexity of osmoregulation in teleost fish.
Chris N. Glover
207
In mammals, the two major circulating corticosteroids are cortisol and

aldosterone. Cortisol is primarily associated with energy metabolism,
whereas aldosterone is mainly involved with the modulation of ion
metabolism. These effects are mediated by two hormone receptors, the
glucocorticoid receptor (GR) that facilitates cortisol action, and the
mineralocorticoid receptor (MR) that promotes aldosterone activity.
Interestingly, the MR has a similar affinity for both cortisol and
aldosterone (Arriza et al., 1987). In fact, given the relative circulating
levels of these hormones, the MR should be bound almost exclusively by
cortisol (Arriza et al., 1987). Specificity of aldosterone response, however,
is governed by the presence of 11>-hydroxysteroid dehydrogenase type 2
(11HSD2), an enzyme that degrades cortisol in aldosterone-responsive
tissues (Funder et al., 1988).
Biochemical and physiological investigations demonstrated what
appeared to be a less sophisticated mechanism of corticoid action in fish.
It is generally assumed that there is no circulating aldosterone in fish (e.g.,
Balm et al., 1989; Sangalang and Uthe, 1994). Instead, it appears that both
glucocorticoid and mineralocorticoid functions in fish are achieved by
cortisol, albeit in conjunction with other endocrine factors (Evans, 2002).
Steroid-binding assays have demonstrated the presence of GR in fish
tissues (Sandor et al., 1984), but molecular evidence was first provided by
Ducouret and colleagues (1995), who cloned the first piscine GR from
rainbow trout. Based on a full-length cloned GR sequence, in vitro
transcription and translation techniques were used to generate the GR.
Results from steroid-binding assays demonstrated a considerably higher
binding affinity for cortisol than aldosterone, functionally characterizing
the receptor as a GR. Furthermore, this receptor was transcriptionally
active in a transfection assay, promoting the expression of a reporter gene
in the presence of a glucocorticoid stimulus. Northern analysis was then
performed that localized the receptor to osmoregulatory tissues.
Subsequently, GR have been fully or partially cloned in other fish species
(Tagawa et al., 1997; Tokuda et al., 1999; Greenwood et al., 2003; Terova
et al., 2005).
Following the initial report, a second group discerned a related GR in
rainbow trout that was a splice variant of the first (rtGR1b; Takeo et al.,
1996). The most significant difference between the initially cloned GR
(rtGR1a) and mammalian GR forms was the presence of an additional 9
amino acid sequence located between the highly conserved zinc fingers
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Fish Osmoregulation
constituting the DNA-binding domain (Ducouret et al., 1995;

Lethimonier et al., 2002). rtGR1b lacked this insert, and thus resembled
mammalian GR. Gene fusion proteins with the native form (rtGR1a) or
with the 9 amino acids deleted (i.e., similar to rtGR1b) were constructed.
A higher constitutive (hormone-independent) expression of the reporter
gene driven by the native GR form was noted. This was attributed to a
higher binding affinity, relative to the insert-deleted GR (Lethimonier
et al., 2002). This difference in activity was noted only when the fusion
proteins were controlled by a single copy of the glucocorticoid responsive
element (GRE) in the promoter region. When constitutive expression was
driven by multiple GREs, there were no differences between the two forms
(Lethimonier et al., 2002). There were also no differences between the two
forms in the presence of hormonal stimulus. This is in contrast to the
findings of Greenwood et al. (2003). These authors reported enhanced
activity of the non-insert form of cichlid GR (i.e., equivalent to rtGR1b).
These differences may be species-dependent. It has been suggested that
this splice variation could be one means of differentially regulating cortisol
function. If GR splice variants activate genes controlled by promoters with
a varying number of GREs in a differential manner, then this could explain
how cortisol specificity is maintained in light of its diverse, pluripotent
actions (Greenwood et al., 2003). These results suggest that molecular
structure is likely to play a significant role in the regulation of cellular
processes under the control of cortisol. This finding may also help explain
why there is still controversy regarding the roles of cortisol in different fish
species (Mommsen et al., 1999).
Given teleost genome duplication, it was of little surprise that a second
GR was cloned from rainbow trout. Using similar methods to those
described above, Bury and colleagues (2003) were able to isolate and
functionally describe rtGR2. This receptor was found to be stimulated by
lower levels of cortisol than rtGR1 and, like the first receptor, no
biologically relevant activity for aldosterone was discerned. The study of
Greenwood and colleagues (2003) identified a homologue to rtGR2 in
cichlid fish. The rtGR2 contains a 4 amino acid insert in the DNA binding
domain, while the cichlid equivalent resembled the mammalian form (no
insert). The different cichlid GRs (the two GR1 splice variants, and the
GR2) were characterized by diverse tissue distributions and activities in
the presence of cortisol (Greenwood et al., 2003).
Chris N. Glover
209
The one hormone-one receptor model of cortisol action was further

revised by the cloning of two receptors from rainbow trout with homology
to mammalian mineralocorticoid receptors (Colombe et al., 2001; Sturm et
al., 2005). These receptors, termed rtMRa and rtMRb, are likely to have
resulted from gene duplication (Sturm et al., 2005). A cichlid MR has also
been cloned (Greenwood et al., 2003). These findings are particularly
curious given the lack of circulating aldosterone in fish. Using steroidbinding assays, the cichlid MR was found to have a high affinity for both
cortisol and aldosterone (Greenwood et al., 2003). In fact, this receptor
had an affinity for cortisol that was 100-fold greater than the GRs, and was
2.5 fold higher that the MR affinity for aldosterone. This is a scenario
similar to that found in mammals (Arriza et al., 1987). In rainbow trout,
aldosterone was favored over cortisol as a ligand (Sturm et al., 2005), again
suggesting species-specificity.
These studies raise significant questions as to the various roles and
regulation of these receptors in fish (see Gilmour, 2005). They also
generate renewed interest into the possible existence of piscine
aldosterone. It is, however, important to note that piscine MRs have yet
to be localized to tissues directly responsible for osmoregulation. Instead,
they appear to be primarily localized to the brain (Greenwood et al., 2003;
Sturm et al., 2005). It is possible that aldosterone may be a local effector
with roles in mediating osmoregulation via modification of central control.
A localized distribution, coupled with the high affinity of the MR for
aldosterone may mean that effective levels of aldosterone elude detection
via common assays. Although bioinformatic searches have failed to detect
an aldosterone synthase in Fugu (Baker, 2003), there appears to be
sufficient evidence to reinvestigate the presence of aldosterone in piscine
tissues. It is also possible that the MR is a ligand for the corticosteroid
precursor 11-deoxycorticosterone, as this molecule also binds rtMR with
high affinity (Sturm et al., 2005). It remains to be determined if this is a
physiological ligand with biologically relevant effects.
The ability of a non-cortisol ligand to mediate its actions via an MR
will likely require a mechanism ensuring the specificity of that ligand. As
described above, in mammalian systems, enzymatic control ensures the
specificity of aldosterone action (Funder et al., 1988). Recent reports have
demonstrated the existence of this enzyme (11HSD2) in fish (Jiang et al.,
2003; Kusakabe et al., 2003). In testes 11HSD2 is responsible for both the
degradation of cortisol and the formation of 11 keto-testosterone, an
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Fish Osmoregulation
important piscine androgen (Kusakabe et al., 2003). 11HSD2 is expressed

in osmoregulatory tissues (Kusakabe et al., 2003). The evidence for a role
of this enzyme in regulation of reproduction is strong. A functional role in
regulating osmoregulation has not yet been explored.
The pluripotent effects of cortisol are likely to be explained by
differential regulation of multiple receptor forms in a species-, tissue- and
cell-specific manner. This highlights the immense underlying complexity
of what was considered a relatively simple system (see Fig. 7.2). Molecular
evidence has begun to unravel the intricate regulation of cortisol functions
in fish, albeit in only a few species and tissues. Nevertheless, these
pioneering studies should provide the structural basis for the expansion of
research into other species.
These recent studies exploring the corticosteroid system in fish have
thus raised more questions than they have provided answers. These
questions have specific relevance for the functioning of osmoregulation in
fish, but also have considerable implications for the evolution of this
system. The ancestral roles of steroid hormones, their regulation, and their
receptors require re-evaluation.
Transgenics and Antisense ApplicationsManipulating
Gene Expression
Techniques that are capable of modifying the expression of genes are of
considerable value in fish biology. Expression-modifying methods include
stable, germline manipulations that are inheritable, such as transgenic and
knockout models. Additionally, methods that target a specific gene or
subset of genes and that are only effective over limited time spans also
exist. As an example of the former category, transgenic growth-enhanced
and freeze-resistant aquacultural species offer considerable potential for
industry (Zbikowska, 2003), while transgenic and knock-out zebrafish are
currently being utilized in developmental studies to great effect (Udvadia
and Linney, 2003). While such inheritable, germline-modified models
offer insight into physiological processes, they currently have limited
applicability and utility for the study of fish osmoregulation. This is due in
part to the likely lethality of gene-knockouts of the well-known
osmoregulatory mediators such as the ion transporters. Furthermore,
because relatively little is known regarding the genomics of other entities
active in osmoregulation, such techniques are unlikely to be cost-effective
Fig 7.2 Schematic diagram illustrating the complexity of corticosteroid regulation of ion transport in teleost fish as elucidated by cellular and
molecular approaches. In mammals (A) a single receptor exists for each of the two circulating hormones. Cortisol will bind to its receptor (white
diamond), and aldosterone to its receptor (white triangle). Specificity is ensured by the presence of the aldosterone-degrading enzyme
11>-hydroxysteroid dehydrogenase type 2 (white circle). Cortisol will drive the expression of genes with glucocorticoid responsive elements
(GREs). In fish (B), there is no convincing evidence for either aldosterone or aldosterone synthase. Multiple isoforms of both glucocorticoid
receptors (white, grey and black diamonds), and mineralocorticoid receptors exist (white and grey triangles), while 11>-hydroxysteroid
dehydrogenase type 2 (white circle) has also been described. Given the lack of aldosterone, it has been suggested that 11-deoxycorticosterone
and 11-deoxycortisol may be relevant mineralocorticoid ligands. Constitutive expression of GRE-driven genes differs depending on the
glucocorticoid receptor that binds, and the number of GREs present. See text for more details.
Chris N. Glover
211
212
Fish Osmoregulation
until suitable targets are identified and sufficient knowledge is acquired

regarding their roles in osmoregulatory processes.
A number of techniques exist for the transient modification of the
expression of single genes. Techniques such as antisense morpholino
oligonucleotides, small interfering RNA (siRNA), and ribozymes are
commonly used to block expression of specific genes in mammals (see
reviews by Kurreck, 2003; Bantounas et al., 2004; Scanlon, 2004), and are
equally applicable in piscine systems. This is a rapidly evolving area of
molecular biology, and ongoing refinements of these methods continue to
improve the specificity and magnitude of gene silencing. One such
modification, with demonstrated application in fish models, is the ability
to temporally control gene inactivation. Caged-RNA can be used to
selectively silence zebrafish genes. Exposure to ultraviolet light, however,
frees these genes for expression, permitting the investigator to switch on
transcription (Ando et al., 2001). This technique could have significant
application for the examination of salinity acclimation, for example. The
capacity to regulate specific genes at specific points of acclimation would
provide detailed knowledge regarding their roles in this process.
Tools capable of modifying expression on a transient basis are
advantageous over germ-line mutations in that they can be performed in
cell culture, and are more cost-effective. Often, however, gene expression
can be controlled only for a limited time, and these techniques are often
most successful in early life stages, when the study of osmoregulatory
function may be of less relevance. These techniques depend on preexisting genetic knowledge, and as such, they are currently favored for use
in fish for which adequate genetic information is available.
Antisense manipulations have been applied to examine specific
cellular functions of developmental genes in the rainbow trout
(Boonanuntanasarn et al., 2002, 2005), zebrafish (Chen and Ekker, 2005),
and medaka (Yamamoto and Suzuki, 2005). The application of such
methods may extend to the control of aquacultural viruses (Alonso et al.,
2005). To date, however, such technologies have not been specifically
applied to examine fish osmoregulation, although as genetic knowledge
regarding the moieties involved expands, so too does the potential for the
use of such gene expression modifications.
Chris N. Glover
213
-omicsMonitoring Global Changes in Cellular

Expression
Global analysis of cellular expression is a powerful molecular tool. The
technological advances in microarray development and analysis of cellular
protein and metabolite profiles have facilitated a recent explosion in the
use of -omic approaches to biological problems. Examination of the
simultaneous expression of thousands of genes, proteins or metabolites
offers obvious benefits over more conventional analyses. We are only
beginning to understand the complicated cellular cascades that enable fish
and other aquatic animals to cope with life in water. Until now, the cellular
and molecular intricacies of osmoregulation have been revealed by studies
concentrating on the measurement of individual enzymes, signalling
molecules, receptors, or a small assemblage of these important entities
(Deane and Woo, 2004). By providing a snapshot of the cellular pathways
that may be activated or deactivated under a given condition, the use of
global expression techniques permits the complex intricacies of
osmoregulation to be elucidated in a holistic manner, potentially defining
novel homeostatic mechanisms. As outlined below, these techniques have
already facilitated several advances in the field of piscine salt and water
balance.
Transcriptomics
The use of physiological model organisms in transcriptomic studies is
restricted by the lack of bioinformatic information for these species. Thus,
while changes in expression profiles of proteins and genes can be
monitored, the identification of these entities is often limiting. There are,
however, transcriptomic approaches which remain amenable. Recent
evidence from ecotoxicological studies in fish employing a model-hopping
approach has provided insight into changes in cellular physiology
(Hogstrand et al., 2002). In these preparations, cDNA derived from the
species of interestin this case rainbow troutwas arrayed against a
cDNA array from a better-characterized species, Fugu. These approaches
are most robust when species are closely related, but can withstand a
certain degree of phylogenetic distance (Renn et al., 2004).
Differential display polymerase chain reaction (PCR) is another
technique that avoids some of the pitfalls associated with a poorly
genetically characterized species. This technique involves the arbitrary
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Fish Osmoregulation
priming of mRNA from both control and treatment groups, with the
subsequent PCR amplification highlighting the presence of unique or
differentially-regulated transcripts (McClelland et al., 1995). This
approach has been the subject of several investigations on fish
osmoregulation. Screening of gill transcripts of eels acclimated to differing
salinities highlighted the presence of an expressed sequence, that on full
cDNA cloning and expression in a cell line, was identified as an inwardly
rectifying potassium channel (Suzuki et al., 1999). Subsequent
immunohistochemical localization to osmoregulatory tissues indicated
that this channel plays a role in seawater acclimation in the eel. Further
studies in a similar vein have also elucidated the importance of cellular
structure changes in euryhalinity, by demonstrating the altered expression
of genes associated with the actin scaffold of chloride cells (Suzuki et al.,
1999; Sakamoto et al., 2002). The discovery of these actin-associated
proteins has since led to significant advances in the cloning and
understanding of factors involved with cell morphology changes during
osmotic challenges (Mistry et al., 2004). These are amongst a number of
transcripts described from such differential display methods that exhibit
differential expression based on salinity (Suzuki et al., 1999; Sakamoto
et al., 2000, 2002; Takeuchi et al., 2000). The role of most of these proteins
is not yet well understood.
Another means of probing global expression differences in non-model
species is suppressive subtractive hybridization (Diatchenko et al., 1996).
This technique involves the annealing of cDNA derived from control
transcripts to that derived from experimental transcripts. Transcripts that
are shared between control and experimental treatments are not amplified
by subsequent PCR amplification. This method has been applied to
examine factors responsible for the acclimation of freshwater tilapia to
seawater (Fiol and Kltz, 2005). Two transcription factors were identified
that were upregulated within hours of transfer. Follow-up investigations
showed protein levels of these factors returned to basal levels within
24 hours and that induction was saline-dependent (Fiol and Kltz, 2005).
These transcription factors are likely to be key regulatory elements in
seawater acclimation.
Perhaps the technique that offers the greatest scope for the discovery
of transcripts involved in osmoregulation is the homologous microarray.
Microarray studies investigating aspects of osmoregulation have been
conducted in plants (Arabidopsis, Satoh et al., 2002) and in the nematode
(Lamitina and Strange, 2003), and have yielded novel information
Chris N. Glover
215
regarding osmoregulation in these species. For example, the former study

discovered the presence of a proline responsive element, by examination
of sequence information derived from proline-inducible transcripts. The
potential utility of such studies suggests that they can make a significant
contribution to investigations of salt and water balance in fish.
The bioinformatics data that facilitates microarray studies is still
generally lacking. However, this information is growing rapidly, and has
advanced to the stage where tools are becoming available for the
investigation of genomics in more physiologically interesting species. For
example, there are now commercially available microarray chips for
Atlantic salmon (Rise et al., 2004). This technology, coupled with the
expansion of bioinformatic data that accompanies such advances,
promises significant potential for the elucidation of osmoregulatory
processes in this, and closely related species.
Methods for exploring changes in gene transcription are all applicable
for examination of aspects of osmotic regulation in fish. In fact, many have
already yielded valuable new information regarding the cellular changes
associated with altered environmental salinity.
Proteomics
Proteomic approaches are perhaps more amenable than transcriptomics
for examining aspects of salt and water balance in fish. This partly reflects
the fact that protein expression levels are a more accurate portrayal of
cellular physiology than are transcript levels. In mammalian liver, for
example, the correlation between mRNA and the corresponding protein
levels was less than 0.5 (Anderson and Seilhamer, 1997). In rainbow trout
hepatocytes, cortisol causes glucocorticoid receptor mRNA levels to
increase, but protein expression levels of glucocorticoid receptor actually
decrease (Sathiyaa and Vijayan, 2003). Similarly, increases in expression
levels of the killifish chloride channel CFTR on seawater exposure are not
matched by increases in CFTR protein (Scott et al., 2004). Consequently,
monitoring changes in protein profile may provide greater functional
insight than genomic approaches.
The technology available for conducting proteomic experiments is
appropriate for research in non-model organisms. Unlike the most robust
genomic methods that require some degree of a priori genetic knowledge,
two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) or
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Fish Osmoregulation
surface enhanced laser desorption/ionization (SELDI) can be performed

without extensive knowledge of the fish proteome. 2D-PAGE is the most
common proteomic approach, and separates proteins in a gel on the basis
of size and isoelectric point. It is the more standard application, and offers
greater scope for determining the identity of unknown proteins. SELDI is
a more recent technological advance, employing time of flight mass
spectrometry with specific assay chip matrices to derive information
regarding a proteins size and some information regarding its
physicochemical properties (Merchant and Weinberger, 2000). It offers
high sensitivity, is perhaps more user-friendly, and has the added
advantage that membrane based proteins are more easily analyzed, thus
offering a greater potential for studies of transport proteins (Jenkins and
Pennington, 2001).
The biggest drawback to using proteomic approaches is that there is
even less bioinformatic data available regarding fish proteins than fish
genes. In the field of fish ecotoxicology, however, it is often enough to
simply state the physical characteristics of unique proteins in a given
exposure condition and therefore proteomic studies are proving of value
for the identification of biomarkers in this field (Hogstrand et al., 2002).
Gel-based proteomic approaches do, however, permit the investigator to
pick proteins of interest and have these sequenced, yielding additional
information regarding the possible identity of the protein. A recent 2DPAGE study in rainbow trout was able to identify nineteen of 23 proteins
excized from the gel, exhibiting the utility of this technique, even in
relatively poorly characterized species (Martin et al., 2003). Advanced
SELDI applications may also include fragmentation analysis, permitting
additional information to be derived from proteins of interest. Both these
technologies hold considerable promise for studies of piscine osmoregulation in the future.
There are, however, examples of how these technologies are being
currently applied to investigate cellular changes that result from osmotic
changes in the environment. Using 2D-PAGE, Kltz (1996) identified
21 proteins that were induced in isolated gill cells of Gillichthys mirabilis
upon hyperosmotic shock and a further 14 that were induced upon hypoosmotic shock. Nine of these proteins were induced in both conditions,
but only three hyperosmotically-induced and five hypoosmoticallyinduced proteins were also induced following thermal shock. The changes
observed were, thus, specific to the type of the challenge, suggesting
Chris N. Glover
217
branchial cells can detect not only changes in the environment but the
nature of these changes, and can respond in an appropriate manner,
independent of higher control.
In a similar study, 2D-PAGE analysis identified nine proteins that were
induced by hypoosmotic shock in gill cells of Gillichthys, but none of these
appeared to share significant homology to any osmotically differentiated
proteins determined in the first study (Kltz and Somero, 1996). This
indicates that important differences may exist between preparations, even
those from the same species. This may result from subtle changes in
experimental methodology, or the physiological state of the animals
themselves. Consequently, the high sensitivity of such techniques may be
both a blessing and a curse, permitting the detection of relatively minor
changes in osmoregulatory entities, but also potentially delineating
changes that are the result of inherent individual variation. Recent studies
have, however, suggested that in general -omic techniques are robust and
offer high reproducibility (Larkin et al., 2005). Nevertheless, careful
consideration of experimental parameters such as sample size is critical
(Wei et al., 2004).
Proteomic approaches can also be adapted for a more targeted
analysis. This was highlighted by a recent study investigating the killifish
cystic fibrosis transmembrane regulator (CFTR) chloride channel (Bridges
et al., 2003). Protein constructs of the C-terminal region of the CFTR
protein were conjugated in a gel column. A lysate of killifish operculum
was passed through the column, and following elution, proteins bound to
the conjugated construct were separated and analyzed via MALDI-TOF.
This elegant approach permitted identification of important accessory
proteins involved in CFTR regulation, and thus discerned the role of actin
organization in this process.
Metabolomics
Metabolomics is, in essence, the assessment of the metabolic substrates
and metabolites that are responsible for, or result from, normal cellular
function (Goodacre et al., 2004). This approach is fast finding favor as a
complementary technique to transcriptomics and proteomics, as it
captures cellular expression at its functional endpoint. Metabolomics is
advantageous from a comparative biology perspective in the sense that the
chemicals analyzed in the metabolome are conserved across all phyla,
meaning that species-specific genetic knowledge is not required for either
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Fish Osmoregulation
conducting or analysing experiments. This implies that, in the short term,

metabolomics has greater application to non-model species, especially
those for which very little genetic knowledge exists, yet are relevant for the
examination of osmoregulation. Furthermore, while the platform for
conducting metabolomic studies is expensive, the per sample running
costs are likely to be less than those for the other global expression analyses
(Goodacre et al., 2004).
A number of different metabolomic platforms exist. Those most
commonly used are nuclear magnetic resonance spectroscopy or mass
spectroscopy, often in combination with a chromatographic technique.
Each platform offers different advantages depending on the number and
nature of metabolites of interest and the required sensitivity (Weckwerth
and Morgenthal, 2005). The ability to detect a given compound depends,
in part, on its standard having being characterized under analytical
conditions equivalent to those under use.
No studies currently exist that have examined the effect of
osmoregulation on the metabolome. The technique has, however, been
applied with success in fish. Viant (2003) demonstrated specific changes
in cellular composition during embryogenesis in the Japanese medaka.
Metabolomic approaches will be best suited to specific subsets of fish
osmoregulation studies. In many cases, the entities of greatest interest in
osmoregulation are the ions, the transporters that facilitate their
movement across epithelia, and the regulatory molecules that control
these processes. Metabolomic approaches are not appropriate for the
examination of these cellular constituents. However, investigation of the
effect of osmoregulation on energy metabolism (Sangiao-Alvarellos et al.,
2005), for example, would benefit significantly from a metabolomic
approach.
Perspectives
The past ten years have witnessed considerable advances in our
understanding of osmoregulation in fish. This has been driven to a large
extent by the development of refined cellular models, and the
indoctrination of molecular techniques. The utilization of these tools has
challenged traditional osmoregulatory dogmas, and has also elucidated the
significant complexity of ionic and osmotic homeostasis. The creation of
novel information has also provided new challenges for researchers in this
discipline.
Chris N. Glover
219
The nature of many of the biological techniques described in this

review is the vast quantities of information they can provide. In particular,
large-scale expression studies and genomic sequencing projects generate
extensive datasets. Such investigations, however, provide only limited
information of gene function. A lesson from the studies already conducted
is that ascribed roles and regulation of osmoregulatory entities in
mammalian systems may not necessarily hold in fish. Consequently, there
is significant scope for the elucidation of functional actions for many of the
structural elements with putative roles in osmoregulation. Cellular models
will provide the impetus for this characterization.
By breaking ionic and osmotic homeostasis down to its basic
constituents and observing them in relatively simplistic model systems,
significant insight into regulatory and functional mechanisms can be
discerned. Effects at this level must, however, be integrated with studies
that examine osmoregulatory processes in intact animals, where neural
and endocrine feedback, and interactions between osmoregulatory
epithelia will alter these processes. Extracellular influences will also be
important. Cardiovascular modification of osmoregulatory surface
perfusion and the roles of mucus as an unstirred water layer, are examples
of tissue-level processes that may have significant influences on ionic
status in fish. These may not be accounted for in cellular and molecular
studies. Equally, consideration should also exist for the influence of
organism-level factors that may impact upon the fish, its cells and
molecules. Behavioral osmoregulation (Lyse et al., 1998), the presence of
ion-disrupting toxicants (Wood, 2001), and switching of energy resources
to reproduction (Le Francois and Blier, 2003), for example, will all have
significant influences.
The cellular tools described herein are well suited for mechanistic
approaches to fish osmoregulation. In association with the structural
information generated by the advances in molecular methods and
physiological data, models of osmoregulatory function are becoming
increasingly refined. The vast diversity of fish, in terms of their genetic
composition, life histories, and habitats is poorly represented by the
relatively few species examined to date. Knowledge of the key
osmoregulatory structures and mechanisms permits testing of
osmoregulatory theories across a far greater range of species, without
requiring access to the whole animal. Molecular and cellular approaches
permit an increasingly developed understanding of the osmoregulatory
minutiae. At the same time, they also facilitate the expansion of
investigations to organisms previously considered impractical for research
220
Fish Osmoregulation
of this nature. These advances have resulted in significant improvements

in our knowledge of ionic and osmotic homeostasis in fish, and will
continue to stimulate and provoke future insights.
Acknowledgements
Drs. Fernando Galvez, Chris Wood and Pl Olsvik are thanked for their
various important contributions to this work.
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CHAPTER
&
Osmoregulation and Fish
Transportation
Paulo Csar Falanghe Carneiro1,
Elisabeth Criscuolo Urbinati2 and Fabiano Bendhack 3, *
OSMOREGULATION
Water is the most abundant constituent of all cells with the exception of
the anhydrobiotic states of extremely few, highly specialized cells. Most
cells have electrolyte (inorganic ion) concentrations accounting for an
osmolality of 200400 mosmol/kg (Wood and Shuttleworth, 1995).
Cellular osmoregulation constitutes a phylogenetically conserved set
of highly complex responses to changes in external osmolality/tonicity to
maintain cell volume, intracellular concentrations of macro- and
micromolecules, protein structure and function, and genomic integrity.
The ubiquitous importance of cellular osmoregulation suggests that a high
and variable environmental salt concentration represents a severe threat
Authors addresses: 1Embrapa Tabuleiros Costeiros. Aracaju, Sergipe, Brazil.
E-mail: paulo@cpatc.embrapa.br
2
Universidade Estadual Paulista. Jaboticabal, So Paulo, Brazil.
E-mail: bethurb@caunesp.unesp.br
3
Pontifcia Universidade Catlica do Paran. Curitiba, Paran, Brazil.
*Corresponding author: E-mail: f.bendhack@pucpr.br
236
Fish Osmoregulation
to the cells. To understand the nature of such a threat, we have to consider

how exactly these cells function. Cell function depends on tightly
controlled ionic and electrostatic interactions between macromolecules,
mainly DNA, RNA, and proteins (Kltz, 2000). In addition, the structure
and function of proteins further depends on water activity and electrolyte
concentration (Hochachka and Somero, 2002). Cell metabolism has been
optimized, during the course of evolution, to function at particular
+
+
concentrations of Na+, Cl, K+, Mg +
2 , Ca 2 , Zn 2 , and other electrolytes
that define intracellular ionic strength. Consequently, any uncontrolled
change in the concentration of these cations interferes with cell
metabolism and function.
The successful establishment of the fish species in different habitats
and environments depends on its ability to cope with salinity differences
between internal (plasma) and external (water) environments through
osmoregulation. Thus, salinity is one of the main environmental factors
exerting a selective pressure on aquatic organisms. The mechanisms
involved result in the maintenance of almost constant or only slightly
variable blood osmolality, over a species-dependent range of salinity.
Below this salinity range, and especially in freshwater, a fish hyperosmoregulates and it is submitted to passive osmotic influx of water and
diffusive loss of ions, mainly Na+ and Cl. Above the isosmotic salinity, the
fish hypoosmoregulates and is exposed to ion invasion and dehydration.
Limiting and compensatory mechanisms include low integument
permeability, active ion uptake in the branchial chambers (mainly gills),
low drinking rate, and production of a high volume of hypotonic urine
(Varsamos et al., 2005).
GILL STRUCTURE AND FUNCTION
Although osmoregulation in fishes is mediated by a suite of structures,
including the gastrointestinal epithelium and kidney, the gill is the major
site of ion movements that balance diffusional gains or losses. The fish gill
is a morphologically and functionally complex tissue that is the site of
numerous, interconnected physiological processes, which are vital to
maintaining systemic homeostasis in the face of changing internal and
external conditions. The features of the gills that enhance gas
exchange also make the gills susceptible to osmotic and ionic movements
between the environment and extracellular fluids, and this condition
necessitates osmoregulation. The branchial epithelium is the primary site
Paulo Csar Falanghe Carneiro et al.
237
of transport processes that counter the effects of osmotic and ionic

gradients, as well as the principal site for pH regulation and nitrogenous
waste excretion. Thus, the branchial epithelium in fishes is a multipurpose
organ that plays a central role in a suite of physiological responses to
environmental and internal changes. The structure and function of the
branchial epithelium as a site of gas exchange dictates that it is also the site
of osmosis and ionic diffusion if gradients exist, and the maintenance of
blood and intracellular tonicity in the face of these osmotic and ionic
gradients across the fish branchial epithelium takes energy (Varsamos
et al., 2005). The fish gill epithelium is composed of several distinct cell
types: pavement cells (> 90%); mitochondrion-rich cells, also called
chloride cells (CCs); and accessory cells, in addition to mucous cells that
are usually on the leading or trailing edge of the filament (Laurent et al.,
1994).
FRESHWATER AND SALTWATER FISHES
Modern freshwater (FW) fishes balance the osmotic uptake of water
through substantial glomerular filtration rates and urine flows and
minimizing renal salt loss by a significant tubular reabsorption of necessary
ions. Net ionic losses in the urine and diffusional outflux across the gill are
balanced by active uptake mechanisms in the gill epithelium plus any ionic
gain from food. Seawater (SW) fishes face the reverse osmotic and ionic
gradients across the branchial epithelium to those found in the freshwater
environment. In teleost saltwater specieswhich are distinctly hypoosmotic to seawaterthe osmotic loss of water is balanced by ingestion of
seawater and subsequent intestinal uptake of NaCl to withdraw needed
water from the lumen contents. The resulting salt load adds to that
produced by the diffusional uptake of NaCl across the gills. The sum is
balanced by gill epithelial NaCl extrusion, because the lack of a loop of
Henle precludes the production of urine that is hyperosmotic to the
plasma (Evans et al., 2005).
The key enzyme to transport processes in the gill and intestine is the
membrane-spanning protein Na+, K+-ATPase. Therefore, the regulation
of Na+, K+-ATPase expression in these organs is of major importance to
fish during SW acclimation. In the opercular membrane of SW-acclimated
fish, chloride cells (CCs) comprise the cellular sites for the excretion of
monovalent ions (Foskett and Scheffey, 1982) and these cells increase in
size and number during the SW acclimation (Laurent and Dunel, 1980).
Even though this cellular function has never been directly demonstrated
238
Fish Osmoregulation
in the gill, there is ample evidence to suggest that CCs have a similar
function in the gills of SW-acclimated fish. On the other hand, the role of
CCs in freshwater FW-acclimated fish is currently being debated (Perry,
1997).
STRESS AND OSMOREGULATION
Stress in fish is a state caused by a stressor that deviates from a normal
resting state. Stress responses are considered a disturbance in the
physiological condition of fish altering their homeostasis and health
(Iwama et al., 1997a).
The primary stress response of fish results in the activation of the
brain-sympathetic-chromafin cells axis (Perry and Reid, 1993) and the
brain-pituitary interrenal axis (Wendelaar Bonga, 1997), leading to the
rapid release of catecholamines and cortisol into the blood stream. The
former event contributes to the initial hyperglycemia and increased
permeability of the surface epithelia, while the latter combines
glucocorticoid and mineralocorticoid actions (Wendelaar Bonga, 1997).
The effects at the level of the gill are particularly important because this
is the site of active ion uptake in freshwater and extrusion in seawater
(Perry and Laurent, 1993), as well as ammonia excretion and gas exchange
(Wilkie, 1997). Generally, when stress disturbs the ability of a fish to
osmoregulate, the ionic composition of its blood comes to resemble that of
the surrounding medium (Pickering, 1993). Stress increases gill
permeability to water and leads to plasma electrolyte changes in a
hyperosmotic or hypoosmotic environment (Cech et al., 1996). Each fish
species regulates the blood fluid concentration within certain limits and
departure from this range is likely to be the result of stress. The point at
which the effect of such influences ceases to be normal and becomes
stressful is difficult to be defined but it is easy to observe the gross effects
of stress. However, by the time these effects on osmotic and ionic
regulation are apparent, the fish may be severely damaged (Eddy, 1981;
McDonald and Milligan, 1997). The osmotic regulation under stress
conditions is extremely expensive and substantial body energy is used to
restore the normal ionic status of the fish. It is widely accepted to possess
an energy-saving effect to rear fish in salinities near their isoosmotic point
(Febry and Lutz, 1987; Gaumet et al., 1995). It was estimated that the
energy required for ion regulation in FW trout gills to be ~1.6% of the
resting metabolic rate and the energy required for ion regulation to be
239
higher in SW gills (5.7%) (Kirschner, 1995). An isolated, saline-perfused

gill arch preparation was used to compare gill energetics in FW- and SWadapted trout. The consumption of FW gills was 33% higher than SW gills.
On a whole animal basis, total gill oxygen consumption in FW and SW
trout accounted for 3.9 and 2.4% of resting metabolic rate, respectively
(Morgan and Iwama, 1999).
STRESS DURING FISH TRANSPORTATION
Stress is present in several steps of fish production, including capture and
transport. Transporting fish is an extremely important part of fish culture.
Live harvested fish may be transported to the market, either to a fishprocessing plant or a fee fishing operation. In both cases, fish must arrive
in good physiological condition and satisfy the criteria of the purchaser.
Especially in the latter case, fish must be able to hook in a very short period
of time after released into the pond (Wurts, 1995). Transportation is a
traumatic procedure that consists of a succession of adverse stimuli,
including initial capture, loading into the transport containers, the actual
transport, unloading and stocking into the new environment (Robertson
et al., 1988). Transport, as any other stressor, may alter the normal
homeostasis of fish and influence the osmoregulation process negatively
(Carneiro and Urbinati, 2001).
Several substances have been used to minimize transport stress as
sodium chloride, calcium sulphate, anesthetics, and others. Sodium
chloride has been used as a mean of stress reduction and increases survival
during transportation of freshwater fish in order to balance with water gain
and electrolytes losses (Carmichael et al., 1984; Carmichael and Tomasso,
1998). The addition of salt to transport water is known to reduce
osmoregulatory disturbances and other physiological responses to stress
factors, besides reducing mortality (Tomasso et al., 1980; Johnson and
Metcalf, 1982; McDonald and Milligan, 1997).
To evaluate the efficiency of salt as a stress reductor, juveniles of
tambaqui Colossoma macropomum, a freshwater species of the Amazon
Basin, were transported in customized plastic boxes in water containing 0,
2, 5 and 8 g of salt/L (Gomes et al., 2003). Plasma cortisol presented a
significant increase after transportation in water either without salt or with
2 g of salt/L, returning to normal levels after 96 hours. The fish exposed
to all salt concentrations had plasma glucose increased after
transportation, except the treatment with 8 g of salt/L of water, returning
240
Fish Osmoregulation
to normal levels within 24 hours. Our studies with matrinx Brycon

cephalus, another Amazonian stenohaline freshwater fish, showed positive
results using common salt during a 4-hour transport (Carneiro and
Urbinati, 2001). Fish transported in water without added salt (control)
showed reduction of the serum sodium levels after transport. Plasma
chloride levels decreased upon arrival in control fish as well as in those
transported in water with 0.1 and 0.3% of added NaCl but not in fish
transported in water with 0.6% of NaCl (Fig. 8.1). During stressful
situations, fish present an increased blood flow and permeability of gills
caused mainly by the action of the catecholamines which facilitate the
exchange of carbonic dioxide for oxygen and improve the supply of the
latter one to support the higher demand for this gas. Nevertheless, any
change in the tegument permeability allows electrolyte loss,
predominantly sodium and chloride, and the water influx, causing serious
osmoregulatory and electrolytic disturbances in freshwater fish. Besides,
sodium chloride has shown positive effect when used during
transportation of several fishes, some species present higher sensibility to
this substance. Gomes et al. (1999) observed increasing mortality and Na+
body levels of jundia fingerlings transported in water containing salt at 0,
1, 3 and 6 g/L.
TRANSPORT WATER
Water quality is also an important factor in determining the success during
fish transportation. Transport of live fish in closed systems results in
significant degradation of water quality throughout the transport period.
Excretory products, mucus and regurgitated food degrade the water quality
and also stress the fish (Berka, 1986).
Total ammonia (NH3 plus NH 4+) is the primary waste product from
the protein metabolism in fish, with un-ionized ammonia being the most
toxic in the hauling water. Within the transportation unit, the main
sources of ammonia are the excretion by fish as a normal part of their
metabolism. As the total ammonia is released into the water, it takes the
form of un-ionized ammonia (NH3), which is highly toxic for fish, and that
of ionized ammonia (NH4+). The main environmental factors affecting the
proportional amount of these two forms, and thus, ammonia toxicity are
the ambient temperature and the pH (Fivelstad et al., 1993).
The increase in carbon dioxide, normally observed during
transportation, causes water pH to decrease. Low pH increases the
proportion of the toxic form of carbon dioxide (CO2), but decreases the
241
Fig. 8.1 Serum sodium (mEq L1) and plasma chloride (mEq L1) of matrinx Brycon
cephalus submitted to transport stress in the presence of added NaCl at the concentrations
0.0 (dotted bars), 0.1 (Dashed bars), 0.3 (vertical lined bars), and 0.6 % (horizontal lined
bars). Different letters indicate differences (P<0.05) among treatments at the same
sampling time. Single and double asterisks indicate differences (P<0.05 and P<0.01,
respectively) between treatments and initial level (shaded bar); all treatments share the
same initial level. Vertical bars represent SEM (N=5). (Carneiro and Urbinati, 2001).
proportion of the toxic form of ammonia (NH3) (Amend et al., 1982).

Carbon dioxide gas behaves as an acid when dissolved in water. It readily
enters the blood plasma as nontoxic bicarbonate ions and renders the
plasma more acidic.
Accumulation of acid in the plasma can have detrimental effects on
the fish osmoregulatory process (Berka, 1986). Low pH impairs the
oxygen-carrying function of haemoglobin. The red blood cells defend their
internal pH against a fall in blood plasma pH using ionic pumps built into
242
Fish Osmoregulation
their cell envelopes. The acid (H+) is removed from the cells in exchange
for Na+. The osmotic pressure rises within and water follows the salt,
causing the red blood cells to swell. This response can be shown clinically
by a rise in the volume of red cells in the blood (measured as the
haematocrit) without a rise in haemoglobin concentration (Fievet et al.,
1988).
Stressful conditions cause passive ion loss and water influx in fish
maintained in hypoosmotic environments, thus negatively affecting the
function of active exchange mechanisms (Wendelaar Bonga, 1997). The
accumulation of ammonia usually observed in transport water may cause
serious problems to the fish, such as increased oxygen consumption and
heart rate, decreased plasma sodium and alteration of the acid-base
balance (Eddy, 1981; Tomasso, 1994; El-Shafey, 1998). Fasting prior to
transport diminishes the amount of food in the digestive tract and reduces
ammonia excretion, being a common practice before transport procedures
of virtually all species or sizes. In spite of NH3 diffusion being the
preferential mechanism of ammonia excretion in teleost, there is a
branchial exchange system between NH4+ and Na+ ions that may become
more important in an environment with sodium ions and high levels of
NH3 (Weirich et al., 1993; Tomasso, 1994). Carneiro and Urbinati (2001)
demonstrated that matrinx transported for 4 hours in water containing
0.6% of added NaCl presented plasma levels of ammonia lower than fish
transported in water containing 0.0 or 0.1% (Fig. 8.2). In fact, fish
submitted to the highest salt concentration first recovered the initial
ammonia level, suggesting the presence of the NH4+/Na+ exchange
system in matrinx.
Calcium is another ion that may be present in water, being essential to
biological processes in fish and linked to osmoregulation, mainly to the
exchange Ca+2/Na+ and cellular permeability (Flik and Verbost, 1993).
The calcium is a low-cost salt, of frequent use in fish transportation,
although its efficiency is yet to be confirmed. The intracellular calcium
concentration of gill cells of a majority of fish species is less than 1 mol.
Nevertheless, even in soft fresh waters with less than 10 mol of this ion,
there is diffusional uptake of Ca+2. The calcium enters to the chloride cells
(CCs) across apical membrane, is transported via cytoplasm and extruded
across the basolateral membrane via Ca+ ATPase or a Na+/Ca+
exchanger (Flik et al., 1995; Evans et al., 2005) (Fig. 8.3).
243
Fig. 8.2 Plasma ammonia (mmol L1) of matrinx submitted to transport stress in the
presence of added NaCl at the concentrations 0.0 (dotted bars), 0.1 (Dashed bars), 0.3
(vertical lined bars), and 0.6% (horizontal lined bars). See Fig. 8.1 for explanation of letters
and asterisks accompanying the value bars (Carneiro and Urbinati, 2001).
Fig. 8.3 Working model for the calcium uptake mechanisms across the freshwater teleost
gill epithelium. (Modified from Evans et al., 2005.)
Teleosts regulate the calcium uptake through the corpuscles of

Stannius that produces stanniocalcin, a hypocalcaemic hormone that
plays an active role in reducing the uptake of this ion in hypercalcic water
(Flik et al., 1995). Besides the prolactin and somatolactin (Kaneko and
244
Fish Osmoregulation
Hirano, 1993), cortisol also influences the regulation of the calcium

uptake, promoting hypercalcaemic effects in fish (Flik and Verbost, 1993).
The ion calcium on water, through anionic reactions, may alter the
hydration of organic structures on gills and thereby reduce their
permeability to both sodium and chloride ions (Potts, 1984), minimizing
ions losses caused by catecholamines action (Wendelaar Bonga, 1997),
normally released into the blood stream during transportation. The ion
calcium presumably also acts on the branchial process of ammonia
excretion by increasing the thickness of mucus layer at the gill surface,
providing a more stable acidic boundary layer in which conversion of NH3
to NH4 would be enhanced, and in which a higher blood to water NH3
gradient maintained (Iwama et al., 1997b). The higher water H+
concentration provided by the mucus layer near the gill epithelium rapidly
ionizes ammonia excreted to NH 4+, contributing to maintain the gradient
difference and the excretion process. In our recent research, the
circulating levels of cortisol increased four times in the control matrinx
and six times in the CaSO4 exposed fish after the packing in the bags. On
the other hand, after transport, fish exposed to calcium presented reduced
levels of cortisol compared to fish not exposed. Also, the excess of calcium
on water induced a decrease of the calcium concentration on blood.
During the recovery period, the lower water calcium concentrations and
the cortisol effect may have stimulated the uptake of this ion, being that
calcitropic action of cortisol become noticeable only in the long term,
approximately 4 days after.
The ions losses and the tendency to water influx are electrolytic
disturbances that can be reduced in freshwater fish transportation
(Carneiro and Urbinati, 2001). Also, in our recent works, the chloride
levels were maintained in calcium-exposed matrinx. Besides, sodium
decreased after transportation (Fig. 8.4). Sodium levels also decreased in
striped bass (Morone saxatilis) transported in water with addition of
100 mg/L of CaCl2 (Mazik et al., 1991).
CONCLUSION
Transport, like any other stressor among the fish culture procedures,
provokes osmoregulatory alterations in fish. Physiological changes might
alter among fish species, but generally all fishes exhibit a similar pattern
concerning hormones releases and electrolyte disturbances, mainly Na+,
Cl and Ca2+. Understanding and previewing these osmoregulatory
245
Fig. 8.4 Serum chloride (mmol/L), sodium (mmol/L) of matrinx transported in water
containing CaSO4 at concentrations of 0 mg/L (dotted bars), 75 mg/L (dashed bars),
150 mg/L (vertical striped bars), 300 mg/L (horizontal striped bars). See Figure 8.1 for
explanation of letters and asterisks accompanying the value bars. Vertical bars represent
SEM (n=8). (Unpublished data).
changes during transportation can help a fish farmer diminish mortality

and loss, as well as increase profit. The use of substances as common salt
and calcium sulphate in the transport water, as well as adopt practices like
handling fish carefully and fasting them before transport are part of the
normal procedures that help fish farmers to cope with osmoregulatory
disturbances caused by transportation.
246
Fish Osmoregulation
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+0)26-4
'
Special Challenges to Teleost
Fish Osmoregulation in
Environmentally Extreme or
Unstable Habitats
Carolina A. Freire* and Viviane Prodocimo
INTRODUCTION
The sea, beyond a certain minimal depth and distance from land, is
actually a very stable environment. However, even if it is environmentally stable, certain oceanic habitats offer special physiological
challenges such as freezing cold water in polar seas or extremely high
pressure in deep seas, which end up interfering with blood hypo-regulation
of marine teleost fish. Some habitats, however, display a wide variation in
Authors address: Departamento de Fisiologia, Setor de Cincias Biolgicas, Universidade
Federal do Paran (UFPR), Centro Politcnico, Bairro Jardim das Amricas, Curitiba, PR,
CEP 81531-990, Brazil.
*Corresponding author: E-mail: cafreire@ufpr.br
This chapter is dedicated to the memory of one of my Ph.D. advisors, Frau Dr Evamaria
Kinne-Saffran. I owe much to her (CAF).
250
Fish Osmoregulation
environmental parameters on a short time scale, imposing the need for

significant and rapid modulation in osmotic homeostasis processes of their
inhabitants. These variable habitats are mainly the estuarine and intertidal
ecosystems, respectively, interfaces between seawater and fresh water, or
between seawater and coastal land. Strictly fresh water bodies may be more
or less stable, depending on their volume and latitudinal location.
However, in temperate latitudes, some may partially freeze in winter, and
in tropical low latitudes some may be either extremely hot or they may
evaporate or present strong acid-based challenges due to intense organic
matter decay.
Approximately 40% of all extant bony fishes (Ostheichthyes) live in
freshwater lakes and rivers, water bodies that cover ~1% of the surface of
the earth, encompassing only ~0.01% of the planets volume of water. The
other 60% of the species live in seawater. Of these, 78% are coastal or
littoral (Nelson, 1994). The oceans cover 70% of the surface of the earth,
and hold 97% of the water volume (Nelson, 1994; Pough et al., 1996).
These numbers reflect the great freshwater diversity of bony fishes. In
addition, both in freshwater and seawater, the largest number of species is
tropical (Nelson, 1994). The bony fishes (the largest taxon of vertebrates)
include the Dipneusti (lungfishes) and Crossopterygii (coelacanth), and
the Brachiopterygii (bichirs), besides the Actinopterygii (ray-finned
fishes). The Actinopterygii are the Chondrostei (sturgeons and
paddlefishes) and the Neopterygii; the latter includes the Lepisosteiformes
(gars), the Amiiformes (bowfins), and the teleosts (Nelson, 1994; Pough
et al., 1996). Teleostean evolution occurred largely in the Mesozoic period,
with all phyletic lineages established in the Cretaceous (Gilbert, 1993).
This chapter will deal only with teleosts, which comprise the vast majority
of bony fish nowadays occupying a wide variety of habitats, and will focus
on showing how some environments challenge fish osmoregulation by
offering extreme values of temperature, pressure, or salinity, or either fast/
steep changes in those parameters, along tidal cycles.
EXTREMELY LOW TEMPERATURES
The special challenges to fish osmoregulation presented by extremely low
temperatures find a natural laboratory in Antarctica. This term has been
used for the intertidal habitat due to its suitability for investigations of the
effect of physical factors on animal physiology and natural distribution
patterns (Hofmann et al., 2002; Tomanek, 2002), but also seems to apply
251
for Antarctica. Seawater in Antarctica displays an average temperature of

1.9C, and it has been that cold for over 10 million years (Eastman, 1993;
Fields and Somero, 1998). The upper 30 m of seawater in Antarctica
contains small ice crystals, and fishes that dwell in superficial waters could
not exist in a super cooled state, due to the abundance of seeding ice
crystals (Eastman, 1993). They must possess an antifreeze protection to
prevent body fluids from freezing. Antifreezers in Antarctic notothenioids
are synthesized in the liver, of molecular weight ranging from 2.6 to 33.7
kDa, consisting of a peptide sequence of variable length with a repeating
unit of the tripeptide Ala-Ala-Thr, with the threonine residue glycosilated
(Eastman, 1993). These antifreeze glycopeptides (AFGP) are present in
the blood of Antarctic notothenioids in higher concentration (35 mg/ml)
than in other body fluids (Eastman, 1993). AFGP exist in higher
concentrations in fishes that remain in shallower ice-laden waters. The
concentration of AFGP decreases with the increase in the depth inhabited
by the fish, as a result of the pressure effect reducing the freezing point of
the water and the reduction until a virtual absence of ice crystals in very
deep waters (Gordon et al., 1962; Eastman, 1993). In fish inhabiting these
deep waters, the amount of AFGP is also dependent on their mode of life:
active species display an even lower content of AFGP than sluggish species
(Whrmann, 1998).
There is a strong association between the presence of AFGP in the
fluids of notothenioids, and the absence of glomeruli in their kidneys. The
secondary loss of glomeruli has been selected as it prevents the loss of
AFGP in the urine; molecules below ~68 kDa pass through the glomerular
barrier (Eastman, 1993). Or, alternatively, the energetic costs of
reabsorption or resynthesis of AFGPs would be huge. Aglomerularism is of
significant adaptive value in the Antarctic environment, and is
apomorphic in notothenioids, as notothenioids of lower latitudes (New
Zealand, Southern South America) lack AFGP and display glomerular
kidneys (Eastman and DeVries, 1986; Eastman, 1993). Interestingly, the
prevention of urinary loss of AFGP is not the sole answer for the condition
of aglomerularism, as it is found in temperate or tropical species, even
freshwater species or marine species that tolerate seawater dilution
(Eastman and DeVries, 1986; Beyenbach and Baustian, 1989; Beyenbach,
1995, 2004; Bone et al., 1995; Baustian et al., 1997). Aglomerular
nephrons produce urine basically by proximal tubular secretion of NaCl,
Mg, and sulphate (Beyenbach and Baustian, 1989; Beyenbach, 1995,
252
Fish Osmoregulation
2004). This is a function shared by proximal tubules of glomerular

nephrons as well, leading to the conclusion drawn by Beyenbach (2004),
that the difference between glomerular and aglomerular urine formation is more
a difference of degree than of kind. The very cold long-standing
temperatures of Antarctica did not result in any significant change in the
renal function of their fish fauna, apart from the loss of glomeruli.
Glomeruli are not the only resource adaptively dismissed in Antarctic
notothenioids. These fishes also lack the heat shock response, expressing
constitutively the normally inducible hsp70 gene, for housekeeping
protein chaperone function during protein synthesis in a cold-denaturing
environment (Place and Hofmann, 2005), but not responding with
upregulation of heat shock proteins upon heat shock (Somero et al., 1998;
Hofmann et al., 2000; Buckley et al., 2004). Being the most stenothermal
fishes of all (Somero et al., 1998; Somero, 2005), they cannot tolerate
water temperature rising to 5-6C, a result of long-time evolution in stably
cold waters (Somero et al., 1998; Somero, 2005). Interestingly, the
stenothermy of Antarctic notothenioids is associated with a high
temperature sensitivity of the Km of their enzymes; eurythermal species
display enzymes with much less temperature-sensitive Kms (Somero et al.,
1998). Antarctic fish never encounter temperature fluctuations in their
environment. This finding highlights the absolute contrast between the
extremely stable Antarctic and the extremely variable estuarine/intertidal
habitats, as will be evident below. Interestingly, as it happens with
aglomerularism, the heat shock response is lost in Antarctic
notothenioids, but is found in New Zealand notothenioids (Hofmann
et al., 2005).
There is a large amount of data on the effect of temperature on
osmoregulation. Temperature strongly influences metabolic rates in
ectotherms such as fish, but the direct effects on osmoregulation may be
very complex (Somero, 2002). The effects of diurnal temperature cycling
as in the intertidal/estuarine habitat (Somero, 2002) may be rather
different from seasonal effects on temperate species subject to freezing in
winter, and also different from long-term evolution under very low
temperature conditions such as in Antarctica, leading to the extreme
stenothermy of Antarctic notothenioids (Somero, 2005). The last two
situations will be discussed in this section of the chapter, and the first
situation (diurnal/tidal variation) will be discussed later in the chapter.
253
Temperature will likely affect ion diffusive rates across membranes,

with probable increase in diffusive fluxes of ions and water upon
temperature increase (Metz et al., 2003), and conversely, an expected
decrease in ion diffusive fluxes upon temperature reduction, including, in
Antarctic fish, a putative special role for the antifreeze peptides, in
blocking ion channels, leading to reduced ion leakage (Prtner et al.,
1998). Differences in acclimation temperatures are often related to
differences in lipid (fatty acid, cholesterol) composition of membranes of
osmoregulatory tissues of fish. In rainbow trout, for example, not only the
degree of unsaturation of fatty acids from membrane lipids, but also their
chain length are temperature-dependent, and cold exposure increases the
incorporation of polyunsaturated fatty acids into phosphatidylserine
(Hazel, 1984; Maffia et al., 1998). These changes in chemical composition
of the membrane lipids upon cold acclimation, in order to ensure proper
barrier function for the membrane have been termed homeoviscous
adaptation (Hazel, 1984), a phenomenon actually first described for
Escherichia coli, under temperature increase (Sinensky, 1974). On their
turn, changes in the lipid microenvironment can affect the activity of
membrane-bound transport enzymes such as the Na,K-ATPase, as
indicated for the intestinal Na,K-ATPase of the gilthead seabream Sparus
aurata (Almansa et al., 2003). Temperature will also directly affect the
activity of the Na,K-ATPase, and while pumps such as the Na,K-ATPase
display a Q10 of 2-4, leak processes are much less temperature sensitive,
with a Q10 of ~1 (Prtner et al., 1998). In some studies, a direct
relationship between temperature of acclimation and specific activity of
the Na,K-ATPase is observed. For example, acclimation of the common
carp Cyprinus carpio to a lower temperature (15C) resulted in lower
activities of the Na,K-ATPase. However, this lower activity was
compensated by increased expression of the enzyme (Metz et al., 2003).
The expression of different isoforms resulting in different enzyme activities
after long periods of acclimation has also been proposed (Metz et al.,
2003). A direct relationship was also observed when Atlantic salmon
(Salmo salar) smolts were transferred to seawater at different temperatures
(2, 4, and 6C); the activities of the branchial Na,K-ATPase (and their rate
of increase along days in seawater) were directly related to the temperature
of acclimation (Arnesen et al., 1998). A positive correlation between
temperature of acclimation and gill Na,K-ATPase activity was also found
for the juvenile turbot Scophthalmus maximus (Imsland et al., 2003). It is
important to add that another parameter associated to temperature may
254
Fish Osmoregulation
actually directly interfere with fish osmoregulation: photoperiod. In the

arctic charr (Salvelinus alpinus), it has been shown that seasonal variation
in seawater tolerance by this species is related to the photoperiod, with
increased seawater tolerance occurring concomitant with extended
periods of daylight in late spring, when the diadromous forms of the species
migrate downstream to feed in coastal waters (Johnsen et al., 2000;
Jrgensen and Arnesen, 2002). The same pattern was observed in the
congener Salvelinus fontinalis (brook charr, Claireaux and Audet, 2000). In
any case, independently of the exact mechanisms mediating the
interaction temperature-osmoregulation, it is clear that mostly for
relatively stenothermal species, either too cold or too hot temperatures,
such as, respectively, 2 and 17C for a salmon smolt (Salmo salar), will
impair osmoregulation (Staurnes et al., 2001).
It should not be forgotten that long-time (over 10 million years)
evolution under constant low temperatures surely led to optimization of
cellular function, cold compensation, under these conditions
(Kunzmann, 1990; Lucassen et al., 2003). To cite a few examples: (1) a
higher amount of unsaturated phospholipids is observed to maintain
membranes fluid next to freezing temperatures (Whrmann, 1998); (2)
cold-adapted protein synthesis machineries display low activation energies
and high RNA translational capacities at similar RNA:protein ratios
(Storch et al., 2005); (3) partial temperature compensation of brain ATPgenerating capacities (Somero et al., 1998); and (4) cold-adapted enzymes
with amino acid substitutions leading to increased flexibility in regions of
the molecule that affect the mobility of structures next to active sites,
causing higher Km values and higher Kcat values at a common temperature
of assay (example, lactate dehydrogenase A4, Fields and Somero, 1998).
The final product of osmoregulatory work comprises the extracellular
concentrations, blood osmolality and ion levels. Thorough investigations
by Arthur DeVries and collaborators in the seventies and eighties have
clearly demonstrated that Antarctic notothenioids display higher blood
osmolality due to elevated NaCl concentrations, when compared to arctic,
temperate/subtropical or tropical fish (Dobbs and DeVries, 1975;
OGrady and DeVries, 1982). This pattern has been confirmed in later
studies (Gonzalez-Cabrera et al., 1995; Romo et al., 2001), and is
summarized in Figure 9.1. Interestingly, arctic seawater fishes (mean
standard error, Na 202 3.1 mM, Cl 185 3.9 mM) display
an intermediary position between the Antarctic (Na 259 4.2 mM,
255
Fig. 9.1 Relationship between sodium and chloride concentrations in plasma or serum in
marine temperate/subtropical/tropical ( ), artic ( ), and Antarctic ( ) teleost fish. Values
plotted for estuarine fish when in full-strength seawater. Diagonal traced line represents
iso-ionic levels of sodium and chloride. Sodium concentrations are higher than respective
chloride concentrations in the same fish, for all environments (P < 0.0001 for arctic and
Antarctic, and P < 0.05 for temperate/subtropical/tropical fishes, paired Students t-test).
Sodium and chloride values for Antarctic fish are higher than values for either arctic or
temperate/subtropical/tropical fish (P < 0.0001 for all comparisons, unpaired Students
t-test), and values for arctic fish are also higher than values for temperate/subtropical/
tropical fish (P< 0.0001 for sodium, and P < 0.001 for chloride, unpaired Students t-test).
Values for temperate and tropical species plotted are from: Eugerres plumieri (PlazaYglesias et al., 1988), Lophius americanus (Beyenbach and Baustian, 1989), Lophius
piscatorius and Pleuronectes flesus (Evans, 1993), Fundulus sp. and Muraena sp. (Bone
et al., 1995), Scophthalmus maximus (Gaumet et al., 1995), Opsanus tau (Baustian et al.,
1997), Sparus sarba (Kelly and Woo, 1999), Mylio macrocephalus (Kelly et al., 1999),
Sphoeroides testudineus (Prodocimo and Freire, 2001), Sparus aurata (Laiz-Carrin et al.,
2005). Values for arctic species plotted are from: Agonus acipenserinus, Anoploploma
fimbria, Atherestes stomias, Daisycottus setiger, Gadus macrocephalus, Hemilepidotus
hemilepidotus, Hemitripterus villosus, Hippoglossus stenolepis, Lepidosetta bilineata,
Lycodes polaris, Malacocottus zonurus, Myoxocephalus polyacanthocephalus, M.
scorpius, Pleurogrammus monopterygius, Theragra chalcogrammus (OGrady and
DeVries, 1982). Due to a wide latitudinal distribution, some species may also be referred
as temperate, instead of polar/arctic. However, they were considered polar, as they have
been collected in the Bering Sea, 56N (OGrady and DeVries, 1982). Values for Antarctic
species plotted are from: Trematomus dendronotus, T. lepidorhinus, T. loenabergii,
Rhigophila dearborni (Dobbs and DeVries, 1975), Dissostichus mawsoni, Gymnodraco
acuticeps, Trematomus borchgrevinki, T. hansoni, T. nicolai (OGrady and DeVries, 1982),
Trematomus bernacchii, T. newnesi (Gonzalez-Cabrera et al., 1995), Notothenia neglecta
(Romo et al., 2001). The names of the species are as cited in the original references.
256
Fish Osmoregulation
Cl 237 5.4 mM) and temperate/subtropical/tropical fish (Na 174

5.1 mM, Cl 154 6.5 mM), as already noted (OGrady and DeVries,
1982), clearly apparent in Figure 9.1, and further confirmed by direct twosample pair wise comparisons (Fig. 9.1). Higher plasma Na than Cl is
apparent in all groups, being a general feature of teleosts, irrespective of
latitude. Why is there an elevated NaCl in blood in Antarctic fish, and
why do arctic fish display this intermediary condition? The reason is
obviously related to water temperatures, as would be expected. In fact,
arctic waters have been cold for a shorter period than Antarctic waters,
and are warmer in summer (OGrady and DeVries, 1982). And
coherently, the Labrador species Myoxocephalus scorpius and Gadus ogac
have displayed increased blood osmolalities in spring, when water
temperatures were of 1.7C, than in summer, when temperatures were of
4-7C (Gordon et al., 1962). This is also consistent with the presence of
antifreeze peptides in the skin, but not in the plasma of boreal fish
(Eastman, 1993). In addition, many of the arctic fish studied by OGrady
and DeVries (1982) reach lower latitudes in their distribution range, being
also considered as temperate fish (www.fishbase.org).
There are at least two possible explanations for greater salt
concentrations in polar than in temperate/subtropical/tropical fish. The
first explanation involves the role of additional osmolytes in freezing
temperature depression of the fluid. It could, however, be argued that
Antarctic fish already have the antifreeze glycopeptides efficiently
protecting their body fluids from freezing (Eastman, 1993). On the other
hand, in fact blood NaCl in Antarctic fish contributes with 40% of the
total freezing point depression (OGrady and DeVries, 1982; Eastman,
1993). The second explanation would be to save energy displaying, for
example, a lower branchial Na,K-ATPase activity, resulting in less salt
secretion and a smaller osmotic gradient to sea water than that maintained
by fishes of milder temperatures. This possibility has been discussed by
Gonzalez-Cabrera and collaborators (1995), who studied the Antarctic
Trematomus bernacchii and T. newnesi, and have observed an enhancement
of the activity of this enzyme in warmer temperatures (4C), when
compared to the activity measured in the normal temperature of their
habitat, 1.5C. This finding and suggestion is consistent with the report
of Prtner and collaborators (Prtner et al., 1998), that evolutionary
adaptation to cold temperatures leads to reduction in the activity of the
Na,K-ATPase: tropical fish display higher activities than temperate fish,
257
which on their turn display higher activities than polar fish. On the other
hand, Ventrella and collaborators (1993) have found enhancement of
Na,K-ATPase activity in osmoregulatory tissues in the long-term coldacclimated sea bass (Dicentrarchus labrax) (Ventrella et al., 1993), and this
result has been reported for other species as well (Prtner et al., 1998),
which could be due to an increased number of enzyme pumps or catalytic
activity of individual transporters. Most probably, both explanations are
complementary. Elevated NaCl leading to a higher blood osmolality in
polar fish indeed aids in the prevention of freezing, and also means energy
savings for the fish, as the maintenance of a lower gradient with respect to
external sea water is indeed less costly, demanding less energy input into
Na,K-ATPase function (OGrady and DeVries, 1982; Eastman, 1993;
Gonzalez-Cabrera et al., 1995; Prtner et al., 1998). However, one must
also consider, as already mentioned above, that the enzymes of Antarctic
fish evolved along millions of years in the very cold and stable
temperatures, having been selected, for instance, for changes in amino
acid sequence, leading to increase in the flexibility of regions involved in
their catalytic conformational changes, supposedly to reduce the enthalpic
energy barriers to conformational changes, increasing catalytic rates in the
cold environment (Fields and Somero, 1998). Furthermore, in vitro values
of Na,K-ATPase specific activity are strongly dependent on the purity of
the membrane microsomal preparation. Crude homogenates lead to much
lower specific activity. Further, in vitro enzyme activities quantified under
optimal conditions reflect the number of functional molecules in the
membrane, and not the actual in vivo activity of the enzyme (Kltz and
Somero, 1995), and may also reflect the isoform(s) expressed under
different acclimation regimes (Gonzalez-Cabrera et al., 1995), as well as
the protein turnover and biosynthetic capacity of the tissue (Prtner et al.,
1998). In addition, all enzymes display a temperature-dependent activity
curve. A comparison of activity as a function of temperature only has
meaning under the same isolation/preparation procedure and with all
other assay parameters kept constant. Some of the parameters have been
considered in the analysis of Prtner and collaborators (1998), but they
have included another variable, mode of life (active/sluggish species) in
their study, which may lead to confusion in the detection of trends
associated to latitudinal temperature; active and sluggish species show
very different metabolic rates under the same conditions (Somero et al.,
1998). Gathering data for a few species, and partially interconverting data
of gill Na,K-ATPase activities obtained in different temperatures, Prtner
258
Fish Osmoregulation
and colleagues (1998) found evidence that evolutionary adaptations to

cold leads to reduced activities, with temperate species being intermediate
between tropical and polar species (Prtner et al., 1998). In conclusion, gill
Na,K-ATPase activities of polar fish may have a trend towards lower values
even at their optimum temperature, when compared to their temperate/
tropical orthologs (Gonzalez-Cabrera et al., 1995; Prtner et al., 1998). On
the other hand, the intestinal Na,K-ATPase specific activity measured at
1C for the Antarctic Trematomus bernacchii was the same as that measured
in the eel Anguilla anguilla at 18C (Maffia et al., 1998). In addition,
temperate fish have been claimed to display lower Na,K-ATPase activities
than tropical fish (Prtner et al., 1998), but their extracellular NaCl levels,
to our knowledge, have never been shown to be distinguishable (Fig. 9.1).
In addition, if the branchial Na,K-ATPase activity of Antarctic fish
increases upon fish acclimation to 4C when compared to 1.5C
(Gonzalez-Cabrera et al., 1995), that may mean that the enzymes of
Antarctic fish do not work at their optimum/maximum activity in the
temperature of the natural habitat of the fish. But, do the enzymes of
temperate/tropical fish work at their maximum in their natural
environmental temperatures? If not, if raising the temperature of the assay
or of the acclimation temperature of the fish raises the activity of their
branchial Na,K-ATPases, does it mean that they are also saving energy in
their normal habitat temperatures? Temperature directly affecting
metabolism and activity is actually a general feature of ectotherms.
Additionally, higher than expected metabolic rates have been measured
for Antarctic fish, again indicating long-term evolutionary adaptation to
this environment (Montgomery and Wells, 1993). In conclusion,
biochemical and physiological cold-adaptation and their effects on
osmoregulation of teleosts is a complex and partially unsettled matter
which can still profit from controlled and broad comparative surveys.
The deep sea is also mostly an extremely stable environment, except
perhaps in the neighborhood of thermal vents, thus with its fish fauna
showing many convergent features with respect to Antarctic fish
(Montgomery and Wells, 1993; Kelly and Yancey, 1999; Yancey et al.,
2002). Bathyal and abyssal fish display elevated levels of trimethylamine
oxide (TMAO) in their tissues, when compared to fish that live in shallow
depths. Teleosts of shallow waters display 10-70 mmol/kg wet weight of
TMAO, while, for example, bathyal teleosts have 103 9, and abyssal
species have 197 2 (Kelly and Yancey, 1999; Yancey et al., 2002). The
259
high content of TMAO is hypothesized to aid in osmoregulation both

directly, by reducing the energetic costs of osmoregulation, and indirectly,
by counteracting deleterious effects of pressure on protein stability (Kelly
and Yancey, 1999; Yancey et al., 2002). Again, the similarity to polar fishes
arises; some of the latter have high glycerol or TMAO (~150 mmol/kg
muscle) concentrations in their tissues (Kelly and Yancey, 1999; Yancey
et al., 2002). Elevated TMAO in polar teleosts may also exert a protective
function, only this time not against high pressure, but against the high
blood NaCl concentrations (Kelly and Yancey, 1999).
EXTREMELY HIGH SALINITIES
What happens in fish that face extreme values of the most important
environmental variable for osmoregulation: salinity? Are there special
mechanisms involved in hyporegulation in hypersaline waters? Or do the
gills of these fish possess a especially powerful salt secretion system,
involving the same mechanism of hyporegulation acting when in fullstrength seawater, but able to establish high salt secretory fluxes and even
steeper salt gradients with respect to ambient salt water? The latter
possibility seems rather more probable. Some euryhaline fish are extremely
tolerant of hypersaline seawater (Fig. 9.2). Amazingly, some incredibly
euryhaline fish that tolerate hypersaline waters are originally freshwater
fish, such as the sailfin molly Poecilia latipinna (Nordlie et al., 1992) (Fig.
9.2), keeping huge osmotic gradients with respect to ambient saline water.
Some species display tolerance to hypersaline media, but actually are not
normally found in hypersaline habitats, an example of which is the
Mozambique tilapia Oreochromis mossambicus. This species has been a
model for strong hyporegulatory capacity in hypersaline medium, but it
naturally inhabits freshwater and brackish water environments in
Southeast Africa. The species has been introduced in other continents and
is now found also in America and Asia, where it also occurs in seawater
(Kltz and Onken, 1993). Morphological studies, using this species in
hypersaline seawater, have demonstrated hyperplasia and hypertrophia of
chloride cells, increasing the number of apical crypts, both in the gill
filament and in the opercular epithelium (Kltz et al., 1995), even forming
chloride cell complexes (Uchida et al., 2000). The same basic result was
found for the euryhaline sea bass Dicentrarchus labrax in 70 ppt seawater,
with the additional record of increased mitochondrial volume and Na,KATPase sites in doubly concentrated seawater (Varsamos et al., 2002). An
260
Fish Osmoregulation
Fig. 9.2 Osmoregulatory homeostasis in euryhaline, freshwater/estuarine ( ), seawater/

estuarine ( ), or diadromous ( , hidden by other symbols) teleost fishes when exposed/
acclimated to variable salinities. Several species were gathered in those categories above;
they may show widely different habits in what concerns their occupation of estuaries. When
more than one time of exposure/acclimation was available in the source study, the most
stable plasma osmolality value, normally for the longest time was chosen. Extremely
euryhaline species for which many salinity values were available, also reaching hypersaline
salinities, were plotted separately. Their symbols are connected by a line, and they are
identified below. Diagonal traced line represents isosmotic values. Values for euryhaline
freshwater/estuarine species plotted are from: Fundulus kansae (Fleming and Stanley,
1965), Eugerres plumieri (Plaza-Yglesias et al., 1988), Salaria fluviatilis (Plaut, 1998),
Oreochromis mossambicus (Uchida et al., 2000), Stizostedion lucioperca (Brown et al.,
2001), Tetraodon nigroviridis (Lin et al., 2004), Oreochromis mossambicus (Cataldi et al.,
2005). Values for euryhaline seawater/estuarine species plotted are from: Blennius pholis
(Davenport and Vahl, 1979), Fundulus heteroclitus (Jacob and Taylor, 1983),
Boleophthalmus boddaerti and Periophthalmus chrysospilos (Chew and Ip, 1990),
Centropomus undecimalis (Prez-Pinzn and Lutz, 1991), Pleuronectes flesus (Evans,
1993), Sparus aurata (Mancera et al., 1993), Scophthalmus maximus (Gaumet et al.,
1995), Fundulus heteroclitus (Zadunaisky et al., 1995), Opsanus tau (Baustian et al., 1997),
Dicentrarchus labrax (Jensen et al., 1998), Salaria pavo (Plaut, 1998), Fundulus
heteroclitus (Marshall et al., 1999), Scophthalmus maximus (Imsland et al., 2003), Sparus
aurata (Laiz-Carrin et al., 2005). Values for diadromous species plotted are from: Anguilla
anguilla (Holmes and Donaldson, 1969), Salmo salar (Arnesen et al., 1998), and Salvelinus
alpinus (Jrgensen and Arnesen, 2002). Data with lines connecting the symbols are
presented, for the freshwater/estuarine fishes Poecilia latipinna (- -, Nordlie et al., 1992)
and Gambusia holbrooki (- -, Nordlie and Mirandi, 1996), the salt marshes inhabitant
Cyprinodon variegatus (- -, Haney and Nordlie, 1997), the tilapia hybrid california
mozambique tilapia from the hypersaline Salton Sea in California, cross between
Oreochromis mossambicus and O. urolepis hornorum (- -, Sardella et al., 2004b), and the
California killifish Fundulus parvipinnis, also found in hypersaline lagoons (- -, Feldmeth
and Waggoner III, 1972). The species Oreochromis mossambicus, Fundulus heteroclitus,
Scophthalmus maximus, and Sparus aurata appear more than once in this figure, as
different studies had different protocols of exposure to different salinities. The names of the
species are as cited in the original references.
261
electrophysiological study using the isolated opercular epithelium of O.

mossambicus acclimated to hypersaline media led to compatible results, of
increased capacity for salt secretion, with a decrease in the leak
conductance resulting from less permeable tight junctions (Kltz and
Onken, 1993).
Although tolerating hypersaline media, some fish display signs of a
high energy cost of osmoregulation under these high salinities, such as
55 ppt for the milkfish Chanos chanos (Swanson, 1998), which actually is
a species naturally found in hypersaline lagoons (of up to 158 ppt salinity)
(Swanson, 1998). A similar result has been observed in the extremely
tolerant gobiid from the Australian desert, which inhabits mound springs
and temporary water bodies around Lake Eyre drainage basin,
Chlamydogobius eremius (Thompson and Withers, 2002). These authors
found increased metabolic rates in gobies maintained in double-strength
seawater (70 ppt) when compared to full-strength seawater (35 ppt).
Freshwater values were the lowest, i.e., there was a direct relationship
between metabolic rate and salinity (Thompson and Withers, 2002),
ascribed to increased energetic cost of osmoregulation in higher salinities.
On the contrary, the euryhaline Mozambique tilapia Oreochromis
mossambicus, caught in brackish water, after 1 month of acclimation to
different salinities, displayed oxygen consumption rates in 1.6 seawater
similar to rates in freshwater, both rates above those measured in full
strength seawater, the experimental salinity closest to the natural salinity
of their environment (Iwama et al., 1997). This U-shaped curves of
oxygen consumption conform to a pattern frequently observed for the
activity of the branchial Na,K-ATPase in euryhaline species (Imsland
et al., 2003). A high Na,K-ATPase activity in either freshwater or seawater
and hypersaline seawater, when compared to brackish water (6-12 ppt) was
observed, for example, in the black seabream Mylio macrocephalus, also
accompanied by an increase in chloride cell numbers in hypersaline media
(50 ppt) (Kelly et al., 1999).
The California Mozambique tilapia is a hybrid of O. mossambicus and
O. urolepis hornorum, and inhabits the hypersaline (43 ppt) Salton Sea in
California. It has been shown to be of similar salinity tolerance as O.
mossambicus (Fig. 9.2), with signs of osmoregulatory stress such as the
increase in apoptotic gill chloride cells, appearing at salinities higher than
55-65 ppt, accompanied by a decrease in oxygen consumption rates
(Sardella et al., 2004b), in a pattern different from the high cost of
262
Fish Osmoregulation
osmoregulation, cited above for Chanos chanos (Swanson, 1998).

Temperature influence is noted, as both low (15C) and high (35C)
temperatures decrease the capacity of the California Mozambique tilapia
to tolerate hypersaline media (Sardella et al., 2004a).
The sheepshead minnow Cyprinodon variegatus is another species that
occurs in hypersaline media (up to 142 ppt) for at least some time (hours
to a few days), inhabiting brackish water coastal salt marshes subject to
intense salinity fluctuations (Haney and Nordlie, 1997). This minnow
shows increased plasma osmolality (Fig. 9.2) and, as the tilapia cited above,
decreased metabolic rates when in higher salinities, above 40 ppt, a
suggestion of reduced energy expenses in hypersaline media, in order to
increase its tolerance time (Haney and Nordlie, 1997). The California
killifish Fundulus parvipinnis is also extremely tolerant of hypersaline
media, naturally occurring in hypersaline lagoons of 55, 87, or even
128 ppt, where its plasma osmolality increased from 350 to 450 mOsm/kg
H2O (Fig. 9.2), with a concomitant decrease in tissue water content
(Feldmeth and Waggoner III, 1972). In conclusion, although metabolic
strategies to deal with extreme high salinity may vary among those rare
species that successfully tolerate and even thrive in such waters, all of
them must be able to produce large outwardly directed salt fluxes through
their branchial epithelia, and possibly reduce inwardly directed diffusive
fluxes (Kltz and Onken, 1993; Kltz et al., 1995; Uchida et al., 2000;
Varsamos et al., 2002). It must be added that transport processes in the
gastrointestinal tract are also potentially upregulated in those species, so
that water gain is in effect, even with such enormous osmotic gradients.
UNSTABLE HABITATS: ESTUARINE AND INTERTIDAL
Rocky intertidal coasts are termed natural laboratories for investigations
on the effects of a changing environment on the physiology of their
inhabitants (Hofmann et al., 2002; Tomanek, 2002; Tomanek and
Helmuth, 2002). This could be extended fairly to estuarine habitats. The
large body of data available on estuarine/intertidal fish physiology in
general, and particularly on fish osmoregulation, can be divided into two
main approaches. The first, more classical approach, detected and
revealed the remarkable euryhalinity and osmoregulatory capacity of these
fish (House, 1963; Evans, 1967; Gordon et al., 1970; Davenport and Vahl,
1979; Bridges, 1993; Prodocimo and Freire, 2001), as well as their
eurythermicity and euryoxic capabilities (Bridges, 1993; Gracey et al.,
263
2001), and even tolerance to emersion, in intertidal amphibious fishes

such as clingfishes (Gordon et al., 1970) and mudskippers (Gordon et al.,
1969, 1978). Regular aerial exposure may lead to a shift from
ammonotelism to ureotelism (Gordon et al., 1970, 1978). A second, more
recent approach, involves the study of their physiological response to the
stress imposed by steep and fast variations in salinity, temperature, and
dissolved oxygen, to highlight the three most important variables
(Dahlhoff, 2004; Hofmann, 2005). These variables interact strongly, e.g.,
salinity and temperature directly affect fish respiration, temperature affects
the metabolic rate with a Q10 of 2-3, salinity affects oxygen solubility and
the diffusion conductance of the gills (Jensen et al., 1993).
The recognition of the natural stress to which the inhabitants of these
ecosystems are regularly exposed led to their appreciation as a natural
laboratory (Hofmann et al., 2002; Tomanek, 2002). Investigating how
these intertidal/estuarine animals respond physiologically to the stress of
their natural habitat makes strong sense, as it integrates the variations in
salinity, temperature, dissolved oxygen, pH, etc. The stress response in fish
has traditionally involved the assessment of blood glucose and cortisol
levels, but more recently, of the expression of regulated heat shock
proteins, the heat shock response (HSR) (reviews in Dahlhoff, 2004;
Hofmann, 2005). The HSR is the regulated expression of the heat-shock
proteins, molecular chaperones whose function is to recognize and bind to
unfolded proteins, preventing their aggregation, and sometimes allowing
their correct refolding (Place and Hofmann, 2001, 2005; Dahlhoff, 2004).
These proteins appear at a higher rate upon temperature, salinity, anoxia,
or chemical stress (Dietz, 1994; Place and Hofmann, 2001; Deane et al.,
2002; Dahlhoff, 2004; Hofmann, 2005). There are many more studies on
temperature activation of heat-shock genes than on salinity activation
(Hofmann, 2005). With salinity, most reports refer to salinity increase,
hyperosmotic shock (Pan et al., 2000), although not exclusively. As would
be expected, salinity reduction is even more common in intertidal or
estuarine habitats, also representing an osmoregulatory challenge to the
animal. However, it may well be that salinity increase more likely leads to
protein denaturation than salinity reduction. Acclimation to both hyper(50 ppt) and hypo-osmotic (6 ppt) salinities for 8 months led to increased
levels of hepatic hsps upon heat shock, in the black seabream Mylio
macrocephalus (Deane et al., 2002).
264
Fish Osmoregulation
Diadromous species face environmental variations, especially in

salinity, although in a different time scale when compared to estuarine/
intertidal species. Species with this habit have been examined with respect
to the HSR as elicited by osmotic/saline stress to isolated tissues: hsp70
was significantly induced in branchial lamellae and hepatic tissue upon
hyperosmotic shock (Smith et al., 1999). A specific connection between
hsps and osmoregulation seems to exist, with complementary parts played
by hsp70 and hsp90 at the onset of seawater adaptation of the salmon (Pan
et al., 2000); hsp70 has more of a chaperone-like function, while hsp90 is
involved in signal transduction regulating salt secretion by the gills (Pan
et al., 2000). A direct linkage between hyperosmotic stress and hsps has
been shown for other systems, and will probably be more clearly revealed
for estuarine and intertidal fish in the near future. Indeed, in the
mammalian kidney, cells of the inner medulla respond to hyperosmotic
stress via TonEBP, the transcription factor tonicity response element
binding protein, which is then translocated to the nucleus, where it can
bind to osmoprotective target genes, such as that of hsp70, and genes
involved with the organic osmolyte response (Kltz, 2005).
Temperature has been largely the most studied variable correlated
with zonation, i.e., vertical distribution of populations in intertidal rocky
shores (review in Somero, 2002). Intertidal fish have been a special focus
of investigation concerning the HSR or activity of heat shock proteins, as
related to the normal temperature fluctuation cycles of their habitat. A
special model has been the marine goby Gillichthys mirabilis, of
extraordinary tolerance to environmental variation (Gracey et al., 2001),
whose major cytosolic molecular chaperone Hsc70 has shown ATPase
activity associated to its chaperoning cycles along the range of
temperatures encountered by the fish in its habitat: 10-35C (Place and
Hofmann, 2001). The plasticity of the HSR response has also been
demonstrated in this gobiid, in that the threshold induction temperature
for the 70-kDa and 90-kDa heat shock proteins (Dietz, 1994), and the
DNA-binding activity of heat-shock factor 1 (HSF1), the transcriptional
regulator of all inducible hsp genes (Buckley and Hofmann, 2002) are not
genetically fixed, but rather respond to the acclimation temperature. A
comparative study has been conducted with sculpins of the genus
Oligocottus: the intertidal sculpin O. maculosus found in upper and lower
tidepools, and its congener, the fluffy sculpin O. snyderi, found only in
lower tidepools during low tide (Nakano and Iwama, 2002). The tidepool
265
sculpin was found to display higher lethal and threshold induction

temperatures for hsp70 and higher constitutive hsp70 levels when
compared to the fluffy sculpin, in accordance with its wider thermal
tolerance (Nakano and Iwama, 2002). Again, the study has been
conducted with respect to temperature fluctuations, but future work
should increase the volume of data on variations in salinity and dissolved
oxygen. Different stressors will not necessarily follow the same pathways,
as has been demonstrated by Kltz (1996) in gill cells of the gobiid G.
mirabilis. The isoforms of the inducible hsp70 were only upregulated by
heat shock, and not by hyperosmotic shock, evidencing the stressor
specificity of the HSR (Kltz, 1996).
Another cellular response intensely activated in these ecosystems is
the antioxidant protection system, largely required after a cycle of hypoxia
during low tide in estuaries and tide pools, followed by re-oxygenation
resulting from the return of seawater with the flow tide (Ross et al., 2001).
It should be pointed out that this recent approach has been mostly applied
to the intertidal habitat, not as much to the estuarine habitat (Ross et al.,
2001), and also, not as much for fish, but mainly for invertebrates and
algae, a fact which coherently reflects the abundance and diversity of the
latter in the intertidal habitat (Abele et al., 1998; Colln and Davison,
1999). Characterization of these responses in intertidal and estuarine
fishes, as a result of variation in salinity, thus in direct relationship to
osmoregulation, is bound to significantly increase in the upcoming years.
Strong euryhalinity and osmoregulatory capacity of intertidal fish has
been shown by Bridges (1993), revising previous data available in the
literature. Euryhaline teleosts, diadromous or with some transit between
estuaries and either fresh- or seawater, or even resident estuarine species,
display very stable plasma osmolalities. In salinities ranging between fresh
water and full strength seawater (35 ppt), the diadromous species
represented in Figure 9.2 had their plasma osmolalities strictly controlled,
between 322 and 377 mOsm/kg H2O, with a mean ( SEM) of 341
9.0 mOsm/kg H2O (Fig. 9.2). Plasma osmolalities of euryhaline species of
marine origin varied between 160 and 410 mOsm/kg H2O (mean SEM:
318 8.5 mOsm/kg H2O), when the fish were exposed to salinities
between fresh water and 50 ppt. The values were vary similar to those
measured for euryhaline species of freshwater origin, in salinities ranging
between fresh water and 63 ppt, and osmolalities between 235 and
414 mOsm/kg H2O (mean SEM: 333 8.2 mOsm/kg H2O). It is
266
Fish Osmoregulation
noticeable that some species have been exposed to salinities representing

extreme of their tolerance range, leading to the breakdown of their
osmotic homeostatic capability. For example, the marine intertidal blenny
Salaria pavo, when in freshwater for 3 months, displays 160 mOsm/kg H2O
of blood osmolality (Plaut, 1998) (Fig. 9.2, marine/estuarine group). On
the other side, the mosquitofish Gambusia holbrooki, after 14 days in 30 ppt
seawater, displays 504 mOsm/kg H2O of blood osmolality (Nordlie and
Mirandi, 1996) (Fig. 9.2). Apart from the mosquitofish, the other species
that show tolerance to hypersaline seawater have yielded very coincident
curves (Fig. 9.2, symbols with lines).
Interestingly, it seems evolutionarily easier for marine fish to move into
freshwater than for freshwater fish to move into the oceans (Nelson,
1994). Marine fish enter the estuary for variable periods of their life cycles
much more often than freshwater fishes. In a very extensive study
conducted on estuarine salt marshes of eastern North America, Nordlie
(2003) has summarized data on 237 species of fishes, and classified them
into one of the following categories: permanent residents, marine nursery
species, diadromous fishes, marine transients, and freshwater transients
(Nordlie, 2003). Marine species that use or live in the estuary for variable
periods add up to 70.0% of those 237 species studied; 52.3% were marine
transients, and 17.7% were marine nursery species. In contrast, freshwater
transients accounted only for 15.2% of the species. A little above nine
percent were permanent residents (9.3%), and only 5.5% were diadromous
(Nordlie, 2003). This fact may have a much more complex explanation
than simply osmoregulation difficulties. But if osmoregulation is at least
part of the reason, this may imply that in general it is less stressful to deal
with hypoosmotic stress than with higher salt, hyperosmotic stress. Water
and salt diffusive fluxes leading at least transiently to extracellular fluid
alterations result in cell volume challenges. And in vitro studies with
animal cells or tissues (not only from fish) show that the capacity for
regulatory volume decrease after hypoosmotic shock is more frequently
found than regulatory volume increase after hyperosmotic shock (Kvers
et al., 1979; Hoffmann and Dunham, 1995). However, there are many
examples of euryhaline fish that can readily switch from salt absorption to
salt secretion, including estuarine species such as the killifish, or
diadromous species (Zadunaisky et al., 1995; Arnesen et al., 1998; Marshall
et al., 1999; Claireaux and Audet, 2000; Johnsen et al., 2000; Lin et al.,
2004). The mechanisms underlying this plasticity have been discussed in
267
other chapters of this volume. Speculatively, the superior number of

marine than freshwater species using estuaries (Nordlie, 2003) may be
related to migratory movements between seawater and fresh water, along
bony fish evolution (Griffith, 1985). Although still a matter of debate,
ostracoderms had a long period of evolution in fresh water; in late Silurian
ostracoderms and early jawed fishes were abundant both in freshwater and
seawater (Pough et al., 1996). Evidences widely accepted as favoring the
hypothesis of a period of evolution in freshwater (or diluted seawater) for
a common vertebrate lineage are the facts that extant fish and vertebrates
in general display low (when compared to nowadays seawater) NaCl
concentrations in their extracellular fluids, and that their kidneys possess
the blood filtering glomeruli (Griffith, 1985; Evans, 1993; Pough et al.,
1996; Vize, 2004). Reduced extracellular salt means less osmotic water
entry in a dilute environment, and glomeruli are ideal structures to
eliminate the water that will have entered osmotically anyway, as
freshwater animals are necessarily hyperosmotic (Vize, 2004). The
contrary view of early vertebrate origin, with evolution and diversification
in the sea, remarks that even the Myxinoidea (hagfishes), a group of
exclusively marine jawless parasite vertebrates (Agnatha), and whose
osmoregulatory strategy is similar to that of marine invertebrates
isosmotic and isoionic (NaCl) with respect to seawaterdisplay glomeruli
in their kidneys (Evans, 1993; Pough et al., 1996). The Myxinoidea have
no features that could be indicative of a freshwater ancestor (Pough et al.,
1996). However, hagfishes could still descend from this putative common
freshwater ostracoderm ancestor lineage, having returned early to the sea
and with a long evolutionary history in the stable and salty depths in the
ocean, having been selected for an osmoregulatory strategy such as that of
invertebrates that never left the sea (Griffith, 1985). Elasmobranchs are
intermediary in the sense that they also display lower NaCl than seawater,
but have developed the strategy of organic osmolyte accumulation (mostly
urea) to be slightly hyperosmotic to seawater, and also always display
glomeruli in their kidneys. Interestingly, there are no examples of
aglomerular elasmobranchs (Vize, 2004). As their strategy promotes
osmotic water entry in seawater, the presence of glomeruli would be
consistent with the availability of sufficient water to promote filtration. As
teleosts face desiccation in seawater, they show the trend for partial or total
morphological loss of glomeruli, or else physiological loss, that is,
glomerular intermittency (Beyenbach, 2004; Vize, 2004). Data on the
268
Fish Osmoregulation
rainbow trout can illustrate this variability in filtering nephrons between

teleosts in freshwater or in seawater. The kidney of the seawater rainbow
trout has only 5% of its total number of glomeruli perfused and filtering,
causing the rainbow trout to be almost functionally aglomerular in
seawater. In contrast, 45% of all glomeruli are perfused and functional in
filtration when the trout is in freshwater (Beyenbach, 2004). Independent
of how concentrated was the water where the first vertebrate evolved, it
is still possible (and tempting to speculate) that the extant lineages of
fishes and other vertebrates derive from a lineage of freshwater
ostracoderms. So, admitting this hypothesis, one may consider that
evolutionarily, marine bony fish descend from a lineage that entered fresh
water from the sea, evolved in diluted waters, and then sometime later
returned to seawater. That is, they have ancestors performing the up- and
downstream migrations, or conversion from hypo- to hyper-regulation of
body fluids and vice-versa. On the other hand, extant freshwater bony fish
(at least some groups) may descend from the first vertebrate migration that
entered fresh water from the sea, possibly ostracoderms. Evolutionarily,
they may have no freshwater to seawater migration in their past, or
evolutionary history. And this ancestry may genetically influence their
ability to turn off salt absorption when the salt content of the water is
increased. This question is fascinating and would be well addressed by a
very broad and comparative analysis of hypo- or hyper-osmoregulatory
capacities among the different orders of bony fish.
CONCLUSIONS
This chapter has reviewed data on teleost fish osmoregulation as
challenged by environments with extreme cold temperatures, high
pressures, or high salinities, or either not as much extreme, but actually
steeply variable coastal habitats such as estuaries and intertidal rocky
shores. Long-time evolution in freezing cold seawater led to increased
blood salt and osmolality, which aids in freezing avoidance, and, as in deep
waters, a protective tissue accumulation of TMAO. Both strategies may
lessen hypoosmoregulatory costs. Very strong hyporegulation in
hypersaline waters is relatively rare, and most likely is the result of a
quantitative (extremely high salt secretory fluxes), rather than a
qualitative difference in the branchial epithelium with respect to regular
full-strength seawater marine hyporegulators. Estuarine and intertidal
habitats challenge teleost fish osmoregulation not only from salinities
269
varying on a short time scale, but also by interactions between variations

in salinity with that of other abiotic parameters such as temperature,
dissolved oxygen, and pH. Estuarine and intertidal teleosts are necessarily
euryhaline, and the effect of the fluctuation in these environmental
parameters is well translated by the stress response pattern of the species
that inhabit such ecosystems.
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+0)26-4
10
Energy Metabolism and Osmotic
Acclimation in Teleost Fish
Jos L. Soengas1,*, Susana Sangiao-Alvarellos1,
Ral Laiz-Carrin2 and Juan M. Mancera2
INTRODUCTION
Euryhaline fish present the capacity to live in different environmental
salinities. The osmolality of the internal milieu in marine teleosts is
equivalent to 10 2% of the environmental salinity (Holmes and
Donaldson, 1969; Maetz, 1974). Most researchers agree on the fact that
water with different ionic and osmotic concentration, with respect to
internal milieu, must impose energetic regulatory costs for active ion
transport: intake in hypotonic environment and uptake in hypertonic
environment. However, there is less agreement concerning the magnitude
of these costs and very little information on the related energetic and
Authors addresses: 1Laboratorio de Fisioloxa Animal, Facultade de Bioloxa, Edificio de
Ciencias Experimentais, Universidade de Vigo, E-36310, Vigo, Spain.
2
Departamento de Biologa, Facultad de Ciencias del Mar y Ambientales, Universidad de
Cdiz, 11510 Puerto Real, Cdiz, Spain.
*Corresponding author: E-mail: jsoengas@uvigo.es
278
Fish Osmoregulation
physiological consequences of life in different salinities (Swanson, 1998;

Boeuf and Payan, 2001). In this way, many studies support the hypothesis
that the energetic cost of ion regulation is lowest in an isotonic with
respect to hypotonic or hypertonic environment, while others did not
support this idea (Morgan and Iwama, 1991; Maxime, 2002). The energy
saved from osmoregulatory processes could be derived to other
physiological processes, such as growth. Thus, a relationship between
environmental salinity and growth has been reported in different
euryhaline fish, but the optimal salinity to obtain the best growth depends
on different factors such as species, temperature, dietary intake, sex, etc.
(Kirschner, 1995; Altinok and Grizzle, 2001; Boeuf and Payan, 2001).
Fish spend large amounts of energy, particularly in the osmoregulatory
organs (i.e., gill, intestine, and kidney), to compensate for these salinity
changes. However, the energetics of these responses to salinity change are
not yet fully understood (Febry and Lutz, 1987; Morgan and Iwama,
1991;Boeuf and Payan, 2001; Brill et al., 2001). This energetic cost can be
estimated based on: (1) the changes observed in the whole body
metabolism using respirometer measurements (McCormick et al., 1989;
Morgan and Iwama, 1991; Kirschner, 1993), or (2) the changes in the
levels of energy substrates and/or in the activities of enzymes controlling
their metabolism. This latter approach has been the one less used
(McCormick and Saunders, 1987) despite of being more advantageous
than the preceding (Febry and Lutz, 1987).
METABOLIC RATES
The cost of osmoregulation in freshwater (FW) and seawater (SW),
according to changes in oxygen consumption, provide very different
estimates ranging from 1% to 20% of the total energy cost of the fish (Febry
and Lutz, 1987; Nordlie et al., 1991; Morgan et al., 1997; Sparks et al.,
2003; Wuenschel et al., 2005). Moreover, those energy costs changed in a
very different way when comparing fish acclimated to different
environmental salinities. Apparently, those different responses are mainly
related to the species assessed.
Accordingly to these data, five patterns of metabolic response to
altered environmental salinities have been suggested by Morgan and
Iwama (1991) in fish including: (1) no change in metabolic rate;
(2) metabolic rate is minimum in isotonic salinity and increases at lower
and higher salinities; (3) metabolic rate increases linearly with salinity;
Jos L. Soengas et al.
279
(4) metabolic rate is higher in freshwater and decreases in isotonic media

(not tolerate seawater); and (5) metabolic rate is highest in seawater
decreasing in other salinities.
CHANGES IN ENERGY METABOLISM DURING OSMOTIC
ACCLIMATION
The capacity of adaptation of euryhaline teleost to changes in
environmental salinity depends on several factors, including the energy
supply and the energy demand necessary for electrolyte shift between
intracellular and extracellular space or the external medium. Successful
salinity acclimation may require a metabolic reorganization in order to
meet the increased energetic demands associated with the exposure to the
new environmental salinity. The metabolic response of teleosts to different
osmotic conditions undoubtedly includes both stress and osmoregulatory
components, but the relative energetic demands of these processes cannot
be discerned from whole animal oxygen consumption (Morgan and Iwama,
1991). Thus, not unexpectedly, alterations in intermediary metabolism
related to osmoregulation are poorly studied in teleosts (Morgan et al.,
1997; Nakano et al., 1998).
In different euryhaline species (Holmes and Donaldson, 1969; Maetz,
1974), including gilthead seabream a species used for our research group
as a model to study the relationship between osmoregulation and
metabolism (Mancera et al., 1993; Laiz-Carrin et al., 2005a; SangiaoAlvarellos et al., 2005), osmoregulatory changes occurring during
acclimation to different osmotic conditions behave normally in two
different stages: (1) an adaptive period with changes in osmotic
parameters; and (2) a chronic regulatory period, where these parameters
again reach homeostasis. However, very few studies have assessed whether
or not the metabolic changes occurring in different tissues during osmotic
acclimation display time courses related to changes in osmoregulatory
parameters.
Therefore, in the following sections, we shall provide a general picture
of the conclusions arrived at from available literature regarding metabolic
changes during acclimation of teleost fish to hyperosmotic and hypoosmotic environments. We will focus these paragraphs in different types of
tissues, such as: (1) tissues involved directly in osmoregulatory work such
as gills, kidney and intestine; (2) tissues involved indirectly in
osmoregulatory work, providing fuels to other tissues such as the case of
280
Fish Osmoregulation
plasma and liver; and (3) tissues theoretically not involved in metabolic
changes occurring during osmotic acclimation such as brain or heart. In
addition, we will also provide information regarding the few cases in which
the time courses of those metabolic changes have been assessed.
Plasma
Osmotic acclimation is known to induce changes in plasma levels of
metabolites such as glucose, lactate, triglycerides or protein (see below).
The assessment of those changes can provide information regarding
metabolic adjustments in the fish for the osmotic acclimation to the new
environment since those metabolites can be used as fuels in
osmoregulatory epithelia.
Increased plasma glucose levels had been observed during acclimation
from FW or brackish water (BW) to SW or hypersaline water (HSW) in
several species of fish such as sea bass (Roche et al., 1989), gilthead
seabream (Sangiao-Alvarellos et al., 2003b; Laiz-Carrin et al., 2005a, b);
rainbow trout (Soengas et al., 1995a), cut-throat trout (Morgan and
Iwama, 1996), cod (Nelson et al., 1996), tilapia (Morgan et al., 1997;
Nakano et al., 1998) or carp (DeBoeck et al., 2000; Yavuzcan-Jildiz and
Kirkaga-Uzbilek, 2001), suggesting a mobilization of glucose to peripheral
tissues in order to satisfy the increased energetic demand observed in
osmoregulatory organs during osmotic acclimation. Moreover, in other
studies using different (coho salmon: Morgan and Iwama, 1998; Atlantic
salmon: Maxime, 2002) or even the same species (tilapia: Morgan et al.
1997, Vijayan et al., 2001), no changes were observed. When euryhaline
fish usually living in SW are acclimated to BW or FW, both decreases such
as in red seabream (Woo and Murat, 1981) or increases such as in tilapia
(Assem and Hanke, 1979), silver seabream (Kelly et al., 1999), and
gilthead seabream (Mancera et al., 1993) have been reported in plasma
glucose levels.
The time course of changes in plasma glucose levels has been assessed
in different species transferred to higher salinity water, showing different
patterns of variation. In rainbow trout transferred from FW to SW, the
increase in plasma glucose was essentially the same throughout the study
(Soengas et al., 1993a). A similar time course has been reported for carp
acclimated from FW to BW (DeBoeck et al., 2000). In contrast, in tilapia
transferred from FW to SW (Nakano et al., 1998) and gilthead seabream
transferred form SW to HSW (Sangiao-Alvarellos et al., 2005), a two-
281
stage behaviour was observed with a sharp increase 1 day after salinity
transfer related to stress followed by a slow decline in the following days
until they reached the previous values.
On the other hand, the time course of changes in plasma glucose is
also available for acclimation to lower salinity water. A two-stage
behaviour has been also reported for sea bass transferred from SW to FW
(Roche et al., 1989) and for gilthead seabream acclimated from SW to BW
(Mancera et al., 1993). Thus, these species showed an increase in glucose
levels in the first stages of acclimation followed by a decline until the
values observed in SW were recovered. However, in another study,
gilthead seabream under the same osmotic transfer displayed two different
peaks (Sangiao-Alvarellos et al., 2005), the first on day 1 and the second
one on days 7 and 14 of the experiment, suggesting an increased
production of glucose.
Plasma lactate levels presented a lineal relationship with
environmental salinity in gilthead seabream (Sangiao-Alvarellos et al.,
2003b; Laiz-Carrin et al., 2005a). This result is comparable with the
decrease observed in red seabream after transfer from SW to diluted SW
(Woo and Murat, 1981), whereas on the other hand, increased plasma
lactate levels were also reported in tilapia after acclimation to SW (Vijayan
et al., 1996). Considering the fact that lactate can be used in tissues like
gills, kidney and brain for their energy requirements (Mommsen, 1984;
Mommsen et al., 1985; Soengas et al., 1998), the rise of plasma lactate
levels observed in parallel with increased salinity suggests that this
metabolite become more important in hyperosmotic conditions,
presumably related to its metabolic use in those organs.
Levels of plasma tryglyceride (TG) have been assessed in several
studies during osmotic acclimation. In gilthead seabream, two clear phases
were distinguished during acclimation to HSW, the first having no
differences with respect to SW-acclimated fish on the first day of
acclimation and the second from day 3 to day 14 with a linear increase in
the levels of this metabolite (Sangiao-Alvarellos et al., 2005). This result
may be in agreement with the enhanced production of TG already
reported in plasma of Atlantic salmon during smoltification (Nordgarden
et al., 2002). Plasma TG levels also increased sharply in the first days of
acclimation to BW of gilthead seabream (Sangiao-Alvarellos et al., 2005)
in agreement with the increase in the amount of plasma lipids in angelfish
transferred to hypoosmotic salinities (Woo and Chung, 1995).
282
Fish Osmoregulation
Plasma protein levels showed different responses to increases in

environmental salinity: (1) increase in cod (Nelson et al., 1996) gilthead
seabream (Sangiao-Alvarellos et al., 2003b; Laiz-Carrin et al., 2005a) and
(2) no changes in red seabream (Woo and Murat, 1981); or (3) decreases
in silver seabream (Kelly and Woo, 1999a). During acclimation of gilthead
seabream to HSW, plasma protein levels described two different phases
with higher levels on the first days and a lower amount at the end of the
acclimation period (Sangiao-Alvarellos et al., 2005). In contrast, Kelly
et al. (1999) did not find any change in this parameter throughout the time
period assessed in black seabream.
Osmotic acclimation may also produce changes in the concentration
of particular amino acids. Thus, changes in osmotic conditions elicited in
tilapia decreased plasma concentration of taurine and glycine (Assem and
Hanke, 1983). A decrease in the concentration of total amino acids in
plasma was observed when fish were acclimated to hyperosmotic
conditions either for angelfish (Woo and Chung, 1995) or tilapia (Vijayan
et al., 1996). However, to our knowledge, the role of amino acid during
osmotic adaptation is not well determined and further studies are required.
Altogether, changes observed in the levels of plasma metabolites
suggested an increased availability of fuels (glucose, lactate and TG),
especially during the first days of acclimation to different osmotic
conditions. These enhanced levels of metabolites would be available to be
used in different tissues. Since osmoregulatory tissues increase their energy
requirements during that process (see below), at least part of those
metabolites could be used to cover their metabolic requirements.
Gills
The gills probably constitute the osmoregulatory organ that consumes the
most energy since they must ensure isosmotic regulation of intracellular
fluid and also anisosmotic regulation of extracellular fluid. Thus, on
transfer of teleost fish into different salinities, sodium and chloride
transport across the gill epithelium switches from ion uptake in hypoosmotic water to ion excretion into hyperosmotic water. Those ion
transport mechanisms involve cotransporters, ion conductive channels,
and ion transport proteins driven by ATP located in the apical and
basolateral membranes of chloride cells and pavement cells (Wilson et al.,
2002). Whereas transport systems and associated ATPases activities have
been described in ample detail (Marshall, 2002; Wilson and Laurent,
283
2002), the biochemical aspects of the mechanisms supplying the required

ATP have been only slightly studied to date (Mommsen, 1984; Perry and
Walsh, 1989). Table 10.1 summarizes the most important changes reported
in literature regarding changes in gill metabolic pathways during osmotic
acclimation in fish.
Fish gills are highly oxidative tissues even in FW, and its oxygen
requirement increases even further when fish are transferred to SW
Table 10.1 Responses in selected metabolic pathways of gills energy metabolism after
acclimation of teleost fish to different osmotic conditions. , increase; , decrease; no
changes.
Pathway
Fish
Acclimation
Response
Use of exogenous
glucose
Rainbow trout
FWSW
Soengas et al. (1995b)
Gilthead seabream
BWSW
SWHSW
BWSW
SWHSW
Sangiao-Alvarellos et al.
(2003b, 2005)
(2003b, 2005)
Laiz-Carrin et al. (2005b)
Soengas et al. (1995b)
LeFranois and Blier
(2004)
(2003b, 2005)
Kelly et al. (1999)
Kelly et al. (1999)
Kelly and Woo (1999b)
Vijayan et al. (2001)
LeFranois et al. (2004)
Kelly et al. (1999)
Kelly et al. (1999)
(2003b, 2005)
Kltz and Jrss (1993)
McCormick et al. (1989)
LeFranois and Blier
(2004)
Leray et al. (1981)
Kltz and Jrss (1993)
Jrss et al. (1983)
Polakof et al. (2006)
Glycogenolysis
Gilthead seabream
Glycolysis
Rainbow trout
Brook charr
FWSW
FWSW
Gilthead seabream
SWHSW
SWBW
Black seabream
SWBW
SWHSW
SWBW
FWSW
BWSW
SWBW
SWHSW
SWBW
BWSW
SWHSW
FWSW
FWSW
FWSW
FWSW
FWSW
FWSW
SWHSW
SWBW
FWSW
SWHSW
SWBW
SWHSW
SWBW
Silver seabream
Tilapia
Wolfish
Pentose phosphate Silver seabream
Black seabream
Gilthead seabream
Aerobic capacity
Tilapia
Atlantic salmon
Brook charr
Amino acid
catabolism
Rainbow trout
Tilapia
Rainbow trout
Gilthead seabream
Lipid catabolism
Masu salmon
Gilthead seabream
Lactate metabolism Gilthead seabream
Reference
Li and Yamada (1992)

284
Fish Osmoregulation
(Vijayan et al., 1996). The energy requirement of the gills is thought to be

maintained by oxidation of glucose and lactate obtained from the
circulation (Mommsen, 1984). Although gill hexokinase (HK) activity
an activity involved in the use of exogenous glucoseis usually low in
teleosts, it is apparently active enough to pace the CO2 release values
observed in that tissue (Mommsen, 1984). This activity increased linearly
with environmental salinity in rainbow trout (Soengas et al., 1995b) and
gilthead seabream (Sangiao-Alvarellos et al., 2003b). Specimens of this
last species acclimated to HSW (Sangiao-Alvarellos et al., 2005) showed
higher gill Na+,K+-ATPase activity and the suspected increased energy
demand of the gills under this osmotic conditions appear to be fuelled at
least by one exogenous fuel such as glucose (as judged by changes in HK
activity). The enhanced levels of plasma glucose at the same time also
suggest this increased use of exogenous glucose by gills (Laiz-Carrin et al.,
2005a; Sangiao-Alvarellos et al., 2005). This indicates an increased energy
demand of gill in the first days after transfer, which is being progressively
declined. From an osmoregulatory point of view, an increased
osmoregulatory work of gills during the first days of acclimation (adaptive
period) seems logical because in this period, the osmoregulatory system is
overactive in order to reach osmotic and ionic homeostasis of internal
milieu (Holmes and Donaldson, 1969; Maetz, 1974).
An enhancement of the glycolytic capacity of gill tissue based on
changes observed in PK activity has been reported in several species
transferred to high salinity environment (Soengas et al., 1995b; Kelly and
Woo 1999a; LeFranois and Blier, 2004; Sangiao-Alvarellos et al., 2005).
Moreover, a decrease of ATP levels in gills along with salinity has been
reported in rainbow trout (Leray et al., 1981), sea bass (Roche et al., 1989)
and gilthead seabream (Sangiao-Alvarellos et al., 2003b). These data
suggest a higher use of ATP in gill of fish adapted to high salinity
environment where an enhancement of gill Na+,K+-ATPase activity is
observed (Laiz-Carrin et al., 2005a; Sangiao-Alvarellos et al., 2005).
On the other hand, the glycolytic capacity of gill tissue has also been
measured during acclimation to BW in several species showing increases
in gilthead seabream (Laiz-Carrin et al., 2005b; Sangiao-Alvarellos et al.,
2005), decreases in black seabream (Kelly et al., 1999) or no changes in
silver seabream (Kelly and Woo, 1999a) when comparing them with SWacclimated fish. This contradictory picture could indicate the existence of
different metabolic pathways for supplying ATP requirements under hypoosmotic conditions in different species.
285
Considering the low amount of glycogen accumulated in gill tissue it

is logical to see no changes in the glycogenolytic potential of this tissue
during osmotic acclimation (Sangiao-Alvarellos et al., 2003b, 2005), since
a raised metabolic demand in gills would not be sufficiently covered by
glycogen mobilization. Since gill tissue is able to oxidize lactate at rates
comparable to those of glucose (Mommsen, 1984; Perry and Walsh, 1989),
and also considering the raise observed in plasma lactate levels in parallel
with increased salinity in several species (see above), an enhanced use of
exogenous lactate through lactate dehydrogenase working in the oxidative
direction cannot be excluded. In this way, preliminary studies carried out
in gilthead seabream also addressed the increase capacity for oxidation of
lactate during acclimation to extreme salinities (Polakof et al.,
unpublished). Finally, another evidence for an increased gill energy
demand in high salinities comes from glucose 6-phosphate dehydrogenase
(G6PDH) activity, which showed a clear decrease in parallel with
increased salinity in tilapia (Kltz and Jrss, 1993) and gilthead seabream
(Sangiao-Alvarellos et al., 2003b). This activity displayed a time course in
which transient increases were observed on the first days after transfer to
HSW or BW. These initial increases coincided with similar rises observed
in tilapia (Kltz and Jrss, 1993), black seabream (Kelly et al., 1999), and
silver seabream (Kelly and Woo, 1999a). Those transient elevations
suggest a process of tissue reorganization consistent with an intermediary
phase of acclimation and coincide in time with the adaptive period
observed in fish after salinity transfer.
To our knowledge there are few studies regarding the impact of protein
metabolism in gills during osmotic acclimation (Jrss et al., 1983; Kultz and
Jurss, 1993) addressing a decreased importance of these metabolites for
fuelling purposes in fish during osmotic acclimation.
Several studies have shown that acclimation from FW to SW produce
an increase in the amount of polyunsaturated fatty acids (PUFA) in gills
of several fish species like rainbow trout (Hansen et al., 1992), masu
salmon (Li and Yamada, 1992), Atlantic salmon (Tocher et al., 2000), sea
bass (Cordier et al., 2002), and eel (Hansen and Grosell, 2004). These
changes cause increased fluidity of the membranes at the time of
hyperosmotic acclimation. Apart from these changes in composition, only
a few studies have assessed changes in lipolytic capacity of gills during
osmotic acclimation addressing an increased capacity (Li and Yamada,
1992).
286
Fish Osmoregulation
Altogether, the results obtained in gills of different fish species lend

support to an increased energy demand in parallel with changes in
environmental salinity as suggested by enhanced Na+,K+-ATPase activity
and glycolytic capacity. This increase in demand appears to be fuelled by
an enhanced use of exogenous glucose as suggested by higher plasma
glucose levels, gills HK activity and levels of glucose and glycogen in gills,
though the possibility of lactate also being used cannot be excluded
(Sangiao-Alvarellos et al., 2003; LeFranois et al., 2004). The
enhancement in energy demand appears to behave in two different stages:
(1) a first one characterized by decreased levels of lactate and glucose; and
(2) a posterior with enhanced use of glucose through glycolysis, pentose
phosphate and glycogenesis on subsequent days of acclimation. These data
suggested an increased energy demand of the tissue in the first days after
transfer, which is progressively declining. From an osmoregulatory point of
view, an enhanced osmoregulatory work of gills during the adaptive period
of acclimation to extreme environmental salinities, thus needing extra fuel
seems reasonable and agrees with the two phases of salinity acclimation
observed in several teleosts (Holmes and Donaldson, 1969; Maetz, 1974;
Mancera et al., 1993, 2002; Laiz-Carrin et al., 2005a; Sangiao-Alvarellos
et al., 2005). Moreover, in those cases where a full range of salinities were
assessed (Kelly and Woo, 1999a; Sangiao-Alvarellos et al., 2003b, 2005;
Laiz-Carrin et al., 2005b), changes appear to be more important during
acclimation to HSW rather than to BW, which may be related to the
higher growth observed in several species when acclimated to BW
compared with those acclimated to SW and HSW.
Kidney
Kidney, in addition to gills, is other important osmoregulatory organ that
plays an active role in the extrusion of divalent ions and elimination of
excess of water in hyperosmotic and hypoosmotic environments,
respectively. Several changes occur in this organ during osmotic
adaptation, including changes in morphology, excretion of divalent ions,
glomerular filtration rate, and urine production (Beyenbach, 1995;
Renfro, 1995). Most of these changes need energy in the form of ATP, and
may be associated with an altered energetic demand that would lead to
changes in kidney intermediary metabolism (McCormick et al., 1989;
Soengas et al., 1994; Kelly and Woo, 1999b). Table 10.2 summarizes the
most important changes reported in literature regarding modifications in
kidney metabolic pathways during osmotic acclimation in fish.
287
Table 10.2 Responses in selected metabolic pathways of kidney energy metabolism after
changes.
Pathway
Fish
Acclimation
Response
Use of exogenous
glucose
Gilthead seabream
SWHSW
BWSW
FWSW
FWSW
SW>HSW
SWBW
SWBW
SWBW
SWHSW
SWHSW
SWBW
SWBW
SWHSW
SWBW
SWHSW
BWSW
FWSW
SWHSW
BWSW
SWHSW
SWBW
SWHSW
SWBW
FWSW
FWSW
FWSW
SWHSW
SWBW
SWHSW
SWBW
SWHSW
SWBW
Glycogenolysis
Glycolysis
Rainbow trout
Rainbow trout
Gilthead seabream
Silver seabream
Gilthead seabream
Black seabream
Glucose export
capacity
Silver seabream
Black seabream
Gilthead seabream
Gluconeogenesis
Rainbow trout
Gilthead seabream
Pentose phosphate Gilthead seabream

Silver seabream
Black seabream
Aerobic capacity
Amino acid
catabolism
Rainbow trout
Atlantic salmon
Rainbow trout
Gilthead seabream
Lipid metabolism
Gilthead seabream
Lactate
metabolism
Gilthead seabream
Reference
(2003b, 2005)
Soengas et al. (1994)
(2003b, 2005)
(2003b, 2005)
Kelly et al. (1999)
Kelly et al. (1999)
Kelly et al. (1999)
Kelly et al. (1999)
(2003b, 2005)
(2003b, 2005)
(2003b, 2005)
Kelly et al. (1999)
Kelly et al. (1999)
McCormick et al. (1989)
Jrss et al. (1983)
Glucose appears to be an important substrate for the kidney in teleosts

based on: (1) the high glycolytic and pentose phosphate shunt capacities
measured in that tissue (Mommsen et al., 1985); and (2) its rates of glucose
use, which are similar to those observed in tissues with important rates
such as brain and gills (Blasco et al., 1996). In Atlantic salmon, the
acclimation to SW decreased citrate synthase and cytochrome oxidase
activities. It led McCormick et al. (1989) to suggest a diminution of
activity of this tissue in higher salinities due to the fact that following SW
adaptation, the teleost kidney produces smaller volumes of a more
concentrated urine than is produced in FW.
288
Fish Osmoregulation
Kidney HK activity showed a sharp decrease in BW- and HSWacclimated gilthead seabream compared with SW-acclimated fish
(Sangiao-Alvarellos et al., 2003b), suggesting that the necessity of kidney
for exogenous glucose was lower in salinities different than usual. The time
course of kidney HK activity has been assessed in this species during
acclimation to HSW, displaying an increase on the first days after transfer
with levels being the highest on day 7 and then returning to normal values
on day 14 (Sangiao-Alvarellos et al., 2005). In contrast, no changes were
noticed in the activity of this enzyme in kidney of rainbow trout during
acclimation to SW (Soengas et al., 1994). On the other hand, during
acclimation of gilthead seabream to BW, a continuous decline from day 1
of experiment onwards was observed (Sangiao-Alvarellos et al., 2005)
suggesting a decreased potential of kidney for using exogenous glucose. In
BW-acclimated fish, a reduction in the activity of the kidney could be
expected because the osmotic and ionic gradients between fish body and
environment are minimal. However, in HSW-acclimated fish, an increased
excretion of ions by kidney could be necessary (Beyenbach, 1995; Renfro,
1995), and thus these metabolic results would lend support for an
enhanced use of another fuel instead of glucose to support the higher
osmoregulatory work of kidney in HSW.
Similar to gill, lactate could also be a good candidate for fuel the
osmoregulatory work of kidney in hyperosmotic and hypoosmotic
environments. Thus, lactate levels increase in this tissue during the
adaptive period of acclimation to HSW in gilthead seabream (SangiaoAlvarellos et al., 2005). On the other hand, a sharp decrease in kidney
lactate levels in BW-acclimated gilthead seabream occurred from day 1 of
acclimation onwards (Sangiao-Alvarellos et al., 2005). This decline,
together with the increased plasma glucose, may suggest that lactate is
increasingly being used as a fuel for kidney in fish adapted to hypoosmotic
environments. In fact, lactate metabolization may be so high that part of
the lactate molecules can be used through gluconeogenesis to enhance
glycogen synthesis, which could match with the increased glycogen levels
observed in kidney of gilthead seabream on days 4 and 7 of acclimation of
gilthead seabream to BW (Sangiao-Alvarellos et al., 2005). In fact,
increased glucose levels can be related to increased conversion rates into
glycogen in kidney and other tissues (Blasco et al., 2001). Moreover, results
obtained by Polakof et al. (2006) also show an increased capacity for lactate
oxidation in kidney of gilthead seabream acclimated to extreme salinities
(BW and HSW).
289
Considering the small amount of endogenous glycogen generally

addressed in fish kidney, this metabolite does not seem important for
fuelling purposes. Accordingly, no changes in glycogen metabolism were
observed during osmotic acclimation in gilthead seabream (SangiaoAlvarellos et al., 2003b, 2005) and silver seabream (Kelly and Woo,
1999b). However, kidney glycogen levels decreased in rainbow trout
acclimated to SW (Soengas et al., 1994). A time course study in gilthead
seabream transferred to higher environmental salinity showed increased
kidney Na+,K+-ATPase activity on the first days after transfer that
coincide with adaptive period described for osmoregulatory system
(Sangiao-Alvarellos et al., 2005). At the same time, changes observed in
the metabolite levels suggest the existence of an increased energy demand
of this tissue on the first days of acclimation in gilthead seabream
(Sangiao-Alvarellos et al., 2005). This energy demand appears to be
reduced on the following days when the fish is in the regulative period. In
HSW-acclimated fish, higher excretion of ions by kidney could be
necessary, and thus metabolic changes observed would lend support for an
increased use of different fuels to support the increased osmoregulatory
work of kidney during the first days of acclimation to HSW.
On the other hand, gilthead seabream showed an enhancement of
glycolysis at the end of the acclimation period to BW in which glucose
levels also decreased and returned to normality (Sangiao-Alvarellos et al.,
2005; Laiz-Carrin et al., 2005b). This would suggest that in the case of
this acclimation, the higher energy requirements are taking place at the
end of the acclimation period. The absence of important changes in the
metabolic parameters assessed may also lend support to the lower
activation of kidney metabolism during the first stages of hypoosmotic
acclimation followed by an increase at the end of the acclimation period.
Further studies are necessary to determine whether or not this pattern of
metabolic variation is a common feature to other euryhaline fish or is
particular to gilthead seabream.
Gastrointestinal Tract
The gastrointestinal tract (GIT) plays an essential role in regulating the
water and electrolyte status of fish (Buddington and Krogdahl, 2004). In
fact, acclimation from FW to SW induces an active absorption of sodium,
chloride at GIT level necessary to drive water passively lost from the body
(Fuentes and Eddy, 1997; Lionetto et al., 2001). In the GIT, there are
290
Fish Osmoregulation
regional differences in the magnitude of electrolyte and water flux due to

differences in the densities and proportions of transporters, ion channels
and permeability. All those processes are energy expensive and, therefore,
changes in energy metabolism of GIT are expected during osmotic
acclimation. However, to our knowdledge, there are very few studies
carried out regarding this issue in literature. The only studies available
report an increased use of amino acids as fuel in rainbow trout acclimated
to SW (Auerswald et al., 1997), decreased triglyceride levels in masu
salmon acclimated from FW to SW (Li and Yamada, 1992), and the fastest
glucose absorption in intestine of rainbow trout acclimated to SW (Brauge
et al., 1994). Moreover, as in in the case of gills, an increased amount of
PUFA has been observed in GIT of different fish during acclimation to
hyperosmotic environments (Hansen et al., 1992; Li and Yamada, 1992).
Liver
The liver is the main site of glycogen/glucose turnover, ammoniogenesis,
fatty acid synthesis, and gluconeogenesis in teleosts (Peragn et al., 1998).
Thus, liver metabolism may be enhanced during osmotic adaptation in
order to make fuels available for metabolic and osmoregulatory processes,
especially in osmoregulatory tissues like gills and kidney (Vijayan et al.,
1996). Table 10.3 summarizes the most important changes reported in
literature regarding metabolic pathways in liver during osmotic
acclimation in fish.
Increased liver glycogenolysis has been usually observed in fish
transferred to hyperosmotic salinities (Hanke, 1991), either in euryhaline
(Assem and Hanke, 1979; Soengas et al., 1995a; Vijayan et al., 1996;
Nakano et al., 1997; Kelly and Woo, 1999b; Sangiao-Alvarellos et al.,
2003b) or in stenohaline species (DeBoeck et al., 2000). However, in other
cases, there were no changes in liver glycogen occurred such as in red
seabream (Woo and Murat, 1981) and rainbow trout (Kroghdahl et al.,
2004). Changes in glycogen levels are generally in agreement with changes
in glycogen phosphorylase (GPase) activity in rainbow trout (Soengas
et al., 1995a), tilapia (Nakano et al., 1997), and gilthead seabream
(Sangiao-Alvarellos et al., 2003b). In contrast, in tilapia acclimated to
HSW, Nakano et al. (1997) failed to address any significant difference in
GPase activity compared with SW-acclimated fish. The mobilization of
liver glycogen would provide glycosyl units ready to be used to fuel
291
Table 10.3 Responses in selected metabolic pathways of liver energy metabolism after
changes.
Pathway
Fish
Acclimation
Response
Glycogenolysis
Tilapia
FWSW
FWSW
SWFW
SWHSW
SWHSW
SWBW
FWSW
FWSW
SWHSW
SWBW
FWSW
FWSW
SWBW
SWBW
FWSW
FWSW
FWSW
SWHSW
SWBW
FWSW
SWBW
SWHSW
SWHSW
SWBW
SWBW
SWHSW
SWBW
FWSW
SWHSW
SWBW
FWSW
FWSW
SWBW
SWBW
SWHSW
FWSW
FWSW
SWHSW
SWBW
Black seabream
Rainbow trout
Gilthead seabream
Glycolysis
Carp
Atlantic salmon
Red seabream
Angelfish
Tilapia
Rainbow trout
Gilthead seabream
Atlantic salmon
Black seabream
Glucose export
capacity
Gluconeogenesis
Gilthead seabream
Angelfish
Black seabream
Red seabream
Tilapia
Gilthead seabream
Pentose phosphate Rainbow trout

Black seabream
Gilthead seabream
Tilapia
Glucose use
Gilthead seabream
Reference
Nakano et al. (1998)
Assem and Hanke (1979)
Nakano et al. (1997)
Kelly et al. (1999)
Kelly et al. (1999)
Soengas et al.
(1993a, 1995a)
Kroghdahl et al. (2004)
(2003b, 2005)
DeBoeck et al. (2000)
Plisetskaya et al. (1994)
Woo and Murat (1981)
Woo and Chung (1995)
Nakano et al. (1997, 1998)
Vijayan et al. (1996, 2001)
Soengas et al. (1995a)
(2003b, 2005)
Kelly et al. (1999)
Kelly et al. (1999)
(2003b, 2005)
Kelly et al. (1999)
Woo and Fung (1981)
Vijayan et al. (1996, 2001)
Sangiao-Alvarellos
et al. (2003b, 2005)
Jrss et al. (1986)
Soengas et al. (1993a)
Kelly et al. (1999)
(2003b, 2005)
Nakano et al. (1997, 1998)
(Table 10.3 contd.)
292
Fish Osmoregulation
(Table 10.3 contd.)
Amino acid
catabolism
Red seabream
Angelfish
Tilapia
Atlantic salmon
Rainbow trout
Gilthead seabream
Lipid catabolism
Coho salmon
Rainbow trout
Tilapia
Gilthead seabream
SWBW
SWBW
FWSW
FWSW
FWSW
FWSW
SWHSW
SWBW
FWSW
FWSW
FWSW
FWSW
FWSW
SWHSW
SWBW

Jrss et al. (1985)
Woo et al. (1978)
Sheridan et al. (1985)
Sheridan (1988)
Brauge et al. (1995)
endogenous pathways such as glycolysis or to be exported to other tissues

that need it (i.e., osmoregulatory organs in fish submitted to salinity
transfer).
The time course of changes in liver glycogen levels in fish transferred
to high salinity environment present different patterns. In gilthead
seabream, a first stage of a sharp decline in glycogen levels followed by a
recovery of levels producing at the end of the acclimation period higher
levels of glycogen in HSW- than in SW-acclimated fish has been observed
(Sangiao-Alvarellos et al., 2005). In other studies, different patterns were
observed, such as: (1) a single phase of continuous decline in rainbow
trout transferred from FW to SW (Soengas et al., 1993a); (2) two phases
in tilapia transferred from FW to SW with no changes at the beginning and
a decline after 2 days (Nakano et al., 1998); and (3) no changes in carp
acclimated from FW to BW (DeBoeck et al., 2000). The enhanced
mobilization of glycogen levels only in the first stage of acclimation to
HSW strongly suggests an increased energy demand from other organs at
that time. Accordingly, the enhancement of glycogenolysis in livers of BWor HSW-acclimated gilthead seabream is accompanied by changes in liver
ATP levels that decreased in parallel with increased salinity (SangiaoAlvarellos et al., 2003b).
On the other hand, during acclimation of gilthead seabream to BW,
liver glycogen levels decreased throughout the experiment suggesting an
enhancement of the energy requirements of this organ through increased
glycogenolysis (Sangiao-Alvarellos et al., 2005). In other species,
acclimation to BW indicated no changes in liver glycogen levels in
angelfish (Woo and Chung, 1995) and black seabream (Kelly et al., 1999).
293
Changes in the glycolytic capacity of the liver were evaluated when

fish were transferred to increased salinities, displaying an enhancement in
that capacity in several species (Soengas et al., 1995a; Vijayan et al., 1996,
2001; Nakano et al., 1997; Kelly and Woo, 1999b; Sangiao-Alvarellos
et al., 2003b). The study of the time course of glycolytic potential in
gilthead seabream transferred from SW to HSW showed an increase of this
capacity only in the first days of acclimation suggesting that was under
those salinity conditions at that time (3 days) the highest energy
requirements of the liver were taking place (Sangiao-Alvarellos et al.,
2003b). Others studies showed an increase in PK activity on the first day
of acclimation of rainbow trout from FW to SW (Soengas et al., 1993a),
and an increase in PFK activity also in the first day of acclimation of tilapia
from FW to SW (Nakano et al., 1998).
On the other hand, acclimation to BW induced in black seabream
(Kelly et al., 1999) and gilthead seabream (Laiz-Carrin et al., 2005b;
Sangiao-Alvarellos et al., 2003b, 2005) a decrease in liver glycolytic
potential. These changes suggest that the mobilization of glycosyl units is
directed towards an increased endogenous use on the first days of
acclimation to BW, whereas on subsequent days, a reduced use is apparent
based on decreased enzyme activities.
The capacity of liver to synthesize glucose through gluconeogenesis do
not appear to be affected by osmotic acclimation in tilapia (Vijayan et al.,
2001) or gilthead seabream (Sangiao-Alvarellos et al., 2003b; Laiz-Carrin
et al., 2005b) in contrast with the increase observed in BW- compared with
SW-acclimated red seabream (Woo and Fung, 1981). However, changes
displayed by glucose 6-phosphatase (G6Pase) activity in gilthead seabream
suggest an increased capacity of liver for exporting glucose on days 1 and
14 in HSW- compared with SW-acclimated fish (Sangiao-Alvarellos et al.,
2003b). No other time courses are available in literature, and the only
studies performed after transfer from SW to HSW address an absence of
changes in liver G6Pase activity in black seabream (Kelly et al., 1999).
Thus, the liver of SW-acclimated fish may have a lower capacity to export
glucose to plasma, which would agree with the finding of decreased liver
G6Pase activity already reported in BW-acclimated fish compared with
SW- and HSW-acclimated fish (Woo and Chung, 1995; SangiaoAlvarellos et al., 2003b, 2005). So, the portion of glucose obtained from
liver mobilization and, therefore, capable of being used in other tissues is
probably higher in HSW- than in SW-acclimated fish in the first stages of
294
Fish Osmoregulation
acclimation. These two stages can be also observed when considering that
the liver of BW-acclimated gilthead seabream may also have a lower
capacity to export glucose to plasma from day 7 of acclimation onwards,
considering the decrease observed in G6Pase activity in gilthead seabream
(Sangiao-Alvarellos et al., 2005). This decrease would agree with the
decrease found at end point in the same enzyme activity in angelfish
acclimated to BW (Woo and Chung, 1995).
Liver glucose 6-phosphate dehydrogenase (G6PDH) activity (an
indicator of potential of the pentose phosphate pathway) is not affected by
hyperosmotic adaptation in most species (Soengas et al., 1993a; Nakano
et al., 1997, 1998; Kelly et al., 1999; Sangiao-Alvarellos et al., 2003b, 2005;
Laiz-Carrin et al., 2005b), whereas increased activity was observed in
rainbow tout transferred from FW to SW (Jrss et al., 1986). In addition,
transfer to hypoosmotic environment induced different behaviour in this
activity: increases in black seabream (Kelly et al., 1999), decreases in
gilthead seabream (Sangiao-Alvarellos et al., 2003b, 2005) or no changes
in red seabream and angelfish (Woo and Murat, 1981; Woo and Chung,
1995).
With respect to amino acid metabolism, no changes have been
observed in hepatic potential for amino acid catabolism during acclimation
of rainbow trout (Jrss et al., 1986), tilapia (Vijayan et al., 2001) and
gilthead seabream (Sangiao-Alvarellos et al., 2003b), suggesting a reduced
importance of these metabolites as fuel in liver during osmotic
acclimation.
In contrast, osmotic acclimation is known to produce mobilization of
liver lipids (Woo et al., 1978) resulting from decreased lipogenesis
(Sheridan et al., 1985; Brauge et al., 1995) and increased lipolysis
(Sheridan, 1988; Li and Yamada, 1992). These results suggest an increased
importance of lipid metabolism in liver during osmotic acclimation,
probably related to the use of those metabolites as fuels to support the
enhanced metabolic work of liver and other tissues observed during
osmotic acclimation. Moreover, fatty acid composition in liver change
during acclimation to SW developed the so-called marine pattern, which
is relatively rich in long-chain PUFA (Li and Yamada, 1992; Tocher et al.,
2000; Cordier et al., 2002), and attributed to the increased capacity of liver
for fatty acid desaturation/elongation observed during osmotic acclimation
(Tocher et al., 2000).
295
Muscle
Fish present two main muscle types: white and red, which generally
behave under anaerobic and aerobic conditions, respectively (Johnston,
1982). White muscle is the main protein accumulating tissue and is
generally used in burst swimming, being principally a glycogen-burning
tissue. In contrast, red muscle is used in sustained swimming, with lipids
being its main fuel (Driedzic and Hochachka, 1978). In addition to the
metabolic changes that take place in muscle directly or indirectly related
to locomotion (Driedzic and Hochachka, 1978), other changes have been
described in processes such as: spawning (Weatherley and Gill, 1987),
feeding (Prez et al., 1988), starvation (Kiessling et al., 1990), stress
(Schwalme and MacKay, 1991) or migration (Leonard and McCormick,
2001). However, changes of muscle metabolism in seawater adaptation
a process which produce an increased energetic demand (Morgan and
Iwama, 1991)have received comparatively little attention.
Only a few studies have assessed metabolic changes in red muscle of
teleost fish during osmotic acclimation. Thus, elevated activities of
enzymes from both the respiratory chain and tricarboxylic acid cycle were
observed in rainbow trout acclimated to SW compared with fish in FW
(Kiessling et al., 1991). Moreover, an increase in the capacity of both
glycogenolysis and glycolysis have been observed in rainbow trout
transferred from FW to SW (Soengas et al., 1995c). The amount of studies
is too low to make clear conclusions but apparently an increased energy
demand arises in this tissue during osmotic acclimation.
In contrast, there are considerably more studies available in literature
regarding metabolic changes in white muscle during osmotic acclimation.
Table 10.4 summarizes the most important results obtained in those
studies. Thus, glycogen levels of fish white muscle present different
patterns of changes during acclimation to increased environmental
salinities: (1) a decrease of glycogen levels (Assem and Hanke, 1979; Woo
and Murat 1981; Claireaux and Dutil, 1992; Soengas et al., 1993b, 1995c);
(2) no changes (Woo et al., 1978; Woo and Fung, 1981); and
(3) contradictory results, i.e., decreases or absence of changes, in muscle
glycogen levels of the same species (Bashamohideen and
Parvatheswararao, 1972; Assem and Hanke, 1979). The production of
glucose from glycogen was enhanced during acclimation of rainbow trout
to SW (Kiessling et al., 1991; Soengas et al., 1993b, 1995c) and during
296
Fish Osmoregulation
Table 10.4 Responses in selected metabolic pathways of white muscle energy

metabolism after acclimation of teleost fish to different osmotic conditions. , increase;
, decrease; no changes.
Pathway
Fish
Acclimation
Response
Glycogenolysis
Red seabream
Tilapia
Cod
SWBW
BWSW
SWFW
SWBW
Rainbow trout
FWSW
Coho salmon
Rainbow trout
FWSW
FWSW
Red seabream
BWSW

Woo and Fung (1981)
Assem and Hanke (1979)
Claureaux and Dutil
(1992)
Soengas et al. (1993b,
1995c)
Woo et al. (1978)
Soengas et al. (1993b,
1995c)
Woo and Fung (1981)
Sea bass
Rainbow trout
Carp
Red seabream
FWBW
FWSW
FWSW
SWBW
BWSW
FWSW
FWSW
FWSW
FWSW
Roche et al. (1989)

Jrss et al. (1985)
Woo and Fung (1981)
Sheridan (1988)
Brauge et al. (1995)
Roche et al. (1989)
Glycolysis
Amino acid
catabolism
Lipid catabolism
Atlantic salmon
Rainbow trout
Sea bass
Carp
Reference
acclimation of carp to BW (DeBoeck et al., 2000). The exogenous source

of glucose was not important, as judged by the absence of an increase in
HK activity in the only study in which this enzyme was measured (Soengas
et al., 1995c).
An increased glycolytic ability related to the increased salinityas
shown by the increased activities of 6-phosphofructo1-kinase, pyruvate
kinase and lactate dehydrogenasewas clearly observed in white muscle
of rainbow trout (Tang and Boutilier, 1991; Soengas et al., 1995c) during
adaptation to seawater and in Atlantic salmon smolts versus parrs
(Leonard and McCormick, 2001). Altogether, these changes suggest an
increased mobilization of glucose obtained from glycogen stores to be
increasingly used through glycolysis in white muscle during osmotic
acclimation.
Of the energy stored as glycogen in white muscle, a very large fraction
can be provided in the form of lactate that besides being reconverted to
297
glycogen in situ (Schulte et al., 1992), it can also be provided to oxidative

tissues via the blood stream (Weber, 1992). Moreover, lactate levels are
known to increase in white muscle and plasma of SW-adapted rainbow
trout (Tang and Boutilier, 1991). Furthermore, the activity of the Cori
cycle (a metabolic pathway related to the transformation of lactate
produced in muscle into glucose in liver) in fish is of minor importance
(Moyes et al., 1992; Schulte et al., 1992), resulting in a poor uptake and
oxidation of lactate in liver. Considering all the above data, we can
hypothesize that an increase in lactate oxidation rates by those tissues able
to use lactate as fuel and involved in osmotic work, such as the gills
(Mommsen, 1984; Perry and Walsh, 1989), may take place during osmotic
acclimation.
As for lipid metabolism, few studies report an enhanced lipolysis in
white muscle during osmotic acclimation of euryhaline fish (Woo and
Murat, 1981; Sheridan, 1988; Brauge et al., 1995), whereas no changes
were observed in many others (Woo and Fung, 1981; Roche et al., 1989;
DeBoeck et al., 2000). Considering the nature of lipid stores in muscle, it
is not surprising to find few changes in this metabolism associated with
osmotic acclimation. Furthermore, several studies addressed an increased
importance of PUFA in the composition of white muscle during osmotic
acclimation (Cordier et al., 2002).
The importance of amino acids for fuelling purposes in muscle during
osmotic acclimation is even lower since in all the studies available in
existing literature, no changes were noticed in amino acid metabolism
during osmotic acclimation (Woo and Fung, 1981; Jrss et al., 1986; Roche
et al., 1989; DeBoeck et al., 2000).
Brain
Brain energy metabolism in fish changes under stress conditions (Soengas
and Aldegunde, 2002), and also after treatment with stress hormones such
as catecholamines (Sangiao-Alvarellos et al., 2003a) and cortisol (LaizCarrin et al., 2002, 2003). In this way, a stressful situation such as
adaptation to different osmotic conditions should produce effects similar
to those of other stressors already evaluated in fish brain. Nevertheless,
this possibility has received little attention to date (Weng et al., 2002).
Weng et al. (2002) addressed for the first time the metabolic changes in fish
brain associated with osmotic adaptation describing changes in ATP levels
and creatine kinase activity in tilapia during the first hours of transfer from
298
Fish Osmoregulation
FW to SW. Besides this study, only a group of studies carried out in gilthead
seabream (Sangiao-Alvarellos et al., 2003b; Laiz-Carrin et al., 2005b;
Polakof et al., 2006) have assessed the metabolic changes in brain during
osmotic acclimation. Those studies demonstrate that acclimation of this
species to salinitieseither lower or higher than normalproduces a
mobilization of brain glycogen levels, which constitutes the major energy
store of fish brain (Soengas and Aldegunde, 2002). The role of this
mobilization is not known but could be related to a stress effect of salinity
on brain metabolism that lead this tissue to activate processes involved
indirectly in the osmoregulatory work.
In addition, the important increase observed in HK activity in gilthead
seabream brain acclimated to extreme salinities compared with those
acclimated to SW (Sangiao-Alvarellos et al., 2003b) reflects the fact that
the necessity of glucose, the main fuel of brain energy metabolism in
teleosts (Soengas et al., 1998), increases under the stress conditions
imposed by the acclimation to extreme salinity environments. The time
course of this increase during acclimation of gilthead seabream to HSW
from day 4 of acclimation onwards suggests that at least part of the
increased glucose within the brain is coming directly from the blood stream
(Sangiao-Alvarellos et al., 2005). Furthermore, HK activity increased in
fish acclimated to BW only at the end of the experimental time. This is
again suggestive of the existence of two stages in the metabolic changes
occurring in this case in the brain after salinity transfer: (1) a first one of
reduced glycogen mobilization and reduced use of exogenous glucose,
followed by (2) a second period of an increased mobilization of glycogen
and potential use of glucose at the end. The enhanced availability of
glucose in this final stage would help to explain the sharp increase in brain
free glucose levels also observed in BW-acclimated fish (SangiaoAlvarellos et al., 2003b, 2005; Laiz-Carrin et al., 2005a).
These changes in glucose phosphorylating capacity in gilthead
seabream are reflected in an increased energy demand based on the high
glycolytic potential observed in BW- and HSW-acclimated fish, as
suggested by changes displayed by PFK activity (Sangiao-Alvarellos et al.,
2003b, 2005). This increased glycolysis may be related to the increase
described by Weng et al. (2002) in both Na+,K+-ATPase and creatine
kinase activities in the brain of tilapia transferred from FW to SW.
Another interesting finding in gilthead seabream was the increase in brain
ATP and lactate levels in parallel with salinity, suggesting that the
299
increased use of carbohydrates is higher than the energy demand of the

brain, resulting in the production of an accumulation of both lactate and
ATP (Sangiao-Alvarellos et al., 2003b). This accumulation also probably
reflects the decreased energy demand of brain at lower salinities since BW
appears to be of a less stressor capacity than HSW. This is in agreement
with the higher growth displayed by gilthead seabream in intermediate
salinity (12 ppt) (Laiz-Carrin et al., 2005b).
The increased availability of glucose within the brain in the first stage
of acclimation in gilthead seabream is apparently not used through
glycolysis or pentose phosphate in BW-transferred fish, since no important
changes were noticed in the activity of selected enzymes from those
pathways, whereas an increased use through glycolysis is apparent in
HSW-transferred fish at that stage (Sangiao-Alvarellos et al., 2005). Also
considering that fish brain appears to use glucose and lactate as fuels
(Soengas et al., 1998), a profound reorganization of brain energy
metabolism is apparently taking place under this situation. Interestingly,
the direction of changes in metabolic parameters in brain of gilthead
seabream is in most cases the same when comparing HSW and BW
acclimation in contrast to the situation described in other tissues
(Sangiao-Alvarellos et al., 2005). This may suggest that the changes
occurring in brain are mainly reflecting a stress salinity response
irrespective of the direction of changes in salinity.
Heart
There are very few studies assessing the changes in heart energy
metabolism during osmotic acclimation. The results provided in those
studies address no changes in glycogen levels of red seabream during
acclimation from SW to BW (Woo and Murat, 1981), a fall in plasma
protein levels in tilapia during acclimation from FW to SW (Venkatachari,
1974), and an increase in the activity of glycolytic and lipolytic enzymes
in Atlantic salmon smolts compared with (Leonard and McCormick,
2001).
Acknowledgements
The authors research has been supported in recent years by grants
BOS2001-4031-C02-01 and VEM2003-20062 (Ministerio de Ciencia y
Tecnologa and FEDER, Spain) AGL2004-08137-C04-03/ACU
(Ministerio de Educacin y Ciencia and FEDER, Spain),
300
Fish Osmoregulation
PGIDT04PXIC31208PN and PGIDIT05PXIC31202PN (Xunta de

Galicia, Spain) to J.L. Soengas, and grant BOS2001-4031-C02-02 and
BFU2004-04439-CO2-01B (Ministerio de Ciencia y Tecnologa and
FEDER, Spain) to J.M. Mancera.
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Fish Osmoregulation
+0)26-4
11
The Renal Contribution to Salt
and Water Balance
INTRODUCTION
This chapter will examine what is currently known about the kidney as it
pertains to salt and water balance within the agnathans (hagfish and
lamprey), elasmobranchs (sharks, rays and skates) and teleosts.
Hagfish are slightly hyperosmotic (1035 mOsm) to their marine
environment (~1000 mOsm) and have achieved this status by retaining
body fluid levels of Na+ and Cl that are similar to concentrations found
in seawater (Hickman and Trump, 1969). For this reason, hagfish are
generally considered to be the only vertebrate group that, like marine
invertebrates, are potentially free from any need for osmoregulation
(Hardisty, 1979; Evans, 1993). However, despite the plasma being at most
2% hyperosmotic to seawater, hagfish extracellular fluid differs from
seawater in almost all the major ions (Hickman and Trump, 1969).
Authors address: Rosenstiel School of Marine and Atmospheric Science, University of Miami,
Miami, Florida, 33149-1098, USA.
E-mail: mcdonald@rsmas.miami.edu
310
Fish Osmoregulation
Specifically, K+ and all of the divalent ions, with the possible exception of
Ca2+, are concentrated in the urine and found in concentrations slightly
below that of seawater in the plasma (Hickman and Trump, 1969; Evans,
1979, 1993; Hardisty, 1979). Na+ appears to be reabsorbed by the kidney,
maintaining plasma levels above seawater. Cl is about the same
concentration in plasma as in urine. The individual regulation of the
plasma ions appears to be the principal function of the hagfish kidney.
However, the hagfish is believed to have the most ineffective of all
vertebrate kidneys; fortunately, its regulatory task is small compared to
osmoregulating organisms (Hickman and Trump, 1969). In contrast,
lampreys, the other surviving agnathan, are osmoregulators and are often
euryhaline as a consequence of their life cycle. In contrast to hagfish,
lampreys have developed the same suite of osmoregulatory mechanisms
that are present in teleost kidneys (see below).
The body fluids of elasmobranchs are similar to agnathans in the sense
that they are almost isoosmotic if not slightly hyperosmotic (~1100
mOsm) to the marine environment (~1000 mOsm; Hickman and Trump,
1969). However, unlike agnathans, their high plasma osmolality is
achieved by combining levels of electrolytes less than that of seawater with
high levels of urea and trimethylamine oxide (TMAO) retained at
concentrations far above those measured in any other vertebrate group.
Consequently, water enters the body by osmosis as it does in freshwater
teleosts and electrolytes enter by diffusion, similar to marine teleosts. The
kidney functions to retain urea and TMAO and eliminate divalent ions
such as Mg2+, SO 42 and PO 42. Excess Na+ and Cl are eliminated by the
rectal gland, which forms a colorless fluid that contains NaCl at nearly
twice its plasma concentration.
Freshwater teleost fish live in an environment that is hypoosmotic
(< 1 mOsm) to their body fluids (280-300 mOsm) and are, consequently,
plagued with a continuous osmotic influx of water and depletion of salts
by diffusion. Active uptake of electrolytes occurs at the gill and the main
responsibilities of the freshwater kidney and urinary bladder are to rid the
body of surplus water while at the same time conserve valuable salts. In
contrast, marine teleosts live in an environment that is hyperosmotic
(~1000 mOsm) to their body fluids (300-320 mOsm). In this
environment, fish constantly gain salt by diffusion and lose water via
osmosis. To compensate, they drink seawater and must desalinize the water
and further modify intestinal fluid so as to promote water absorption.
311
Marine fish actively excrete most of their monovalent ions via the gill and
while the gill extrudes Na+ and Cl, the kidney and urinary bladder serve
to conserve water but excrete Mg2+, SO 42 and other divalent ions.
Kidney Morphology
The hagfish kidney is quite simple, with segmentally arranged glomeruli
draining into short neck segments and then into paired common
archinephric ducts that have some of the structural and functional
attributes of proximal tubule I of other fishes and tetrapods (Fig. 11.1A;
Evans, 1993). The glomeruli in hagfish are supplied by branches of
segmental arteries arising from the dorsal aorta (Hickman and Trump,
1969). The capillary network of the ureters is served by the postglomerular
circulation and by arteries directly from the aorta; unlike teleosts, there is
no portal circulation (Hickman and Trump, 1969). In comparsion,
lampreys have a much more developed kidney, with distinct glomeruli,
proximal tubule I, distal tubule and collecting duct (Fig. 11.1B; Hentschel
and Elger, 1989; Evans, 1993). The proximal and distal tubules are
arranged in a loop, reminiscent of the loop of Henle of the mammalian
kidney and the elasmobranch kidney (see below). Lampreys also lack a
renal portal system which, in teleosts, enables tubular secretion to
continue in the absence of glomerular perfusion (Brown and Rankin,
1999). Because of this difference in renal circulation, agnathans and
teleosts regulate their GFR differently.
The internal anatomy of the elasmobranch kidney is complex but
highly organized. In general, the marine elasmobranch kidney consists of
a large glomerulus, proximal tubule I and II, a countercurrent loop system
connecting the proximal and distal tubule and the collecting duct system
(Fig. 11.1C). In general, the kidneys are divided into two regions, a sinus
(ventral) zone and the bundle (dorsal) zone that is enclosed by a
peritubular sheath (Lacy et al., 1985; Lacy and Reale, 1986; Friedman and
Hebert, 1990). A single nephron has two highly coiled loops that enter the
sinus zone, which is richly endowed with blood vessels and is a region of
a high rate of blood flow. There appears to be no organized pattern to the
loops in the sinus zone. In contrast, five lengths of the same nephron are
arranged to lie in parallel within the bundle zone. The segments pass
through this region and form convoluted loops within the bundle zone and
dip into the sinus zone, allowing the tubule fluid to pass twice through
each of the two zones. The physiological significance of this behavior is not
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Fish Osmoregulation
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Fig. 11.1 Schematic representation of nephron structure and its relative importance to
osmoregulation within (A) hagfish (B) lamprey, (C) marine elasmobranchs (D) freshwater
elasmobranchs (E) freshwater teleosts, (F) marine glomerular teleosts and (G) marine
aglomerular teleosts. Adapted from Hickman and Trump (1969) and Evans (1993).
entirely clear but it very likely facilitates the conservation of organic

solutes in the plasma, namely urea (Braun and Dantzler, 1997).
Interestingly, the freshwater elasmobranch kidney (Fig. 11.1D) lacks the
nephron loops observed in marine elasmobranchs.
The teleost kidney is much less complex than that of the
elasmobranch and, in some ways, the lamprey. Freshwater teleost fish have
a kidney nephron that includes a glomerulus, proximal tubule I and II,
distal tubule and a collecting tubule and duct (Fig. 11.1E; Nishimura and
Imai, 1982; Hentschel and Elger, 1989; Evans, 1993). The kidney of
marine teleosts is morphologically reduced in comparison. In marine
teleosts, various stages of glomerular degeneration is observed, an
adaptation that reaches its pinnacle in approximately 30 fish species that
are aglomerular (i.e., without a glomerulus; Fig. 11.1F, G). Proximal tubule
I is also missing in aglomerular species and the distal tubule is usually
313
lacking in both glomerular and aglomerular marine fish. Unlike freshwater

teleosts, the proximal segment(s) in marine teleosts connect directly to the
collecting duct via the collecting tubule. In contrast to elasmobranchs and
lamprey, the teleost kidney lacks any loop formations within the tubule. In
general, arterial blood to the kidney in teleosts is supplied by renal arteries
arising from the dorsal aorta or by renal branches from segmental arteries
(Forster, 1953; Hickman and Trump, 1969; Nishimura and Imai, 1982;
Braun and Dantzler, 1997). In glomerular forms, these arteries give rise to
afferent arterioles which supply the glomerular capillaries and then drain
into efferent arterioles, which break up into a network of sinusoids and
peritubular capillaries (Forster, 1953; Hickman and Trump, 1969). Thus,
in most teleost fish, the glomerular blood supply is arterial while the renal
tubules have a double supply of blood, the first from the efferent
glomerular arterioles and a second from the venous renal-portal system. In
marine aglomerular teleosts, the latter is the only means of blood flow to
the kidney and in marine fish in general, the venous supply becomes more
and more important, depending on the extent of glomerular degeneration.
Essentially, in the absence of glomeruli and in the presence of low filtration
rates in glomerular marine fish, renal functions rely largely on the venous
perfusion that delivers blood to the peritubular sinuses in the kidney
(Beyenbach, 2004).
Osmoregulatory Processes of the Kidney
i. Glomerular filtration
The first step in renal osmoregulation is the filtration of the plasma by the
glomerulus. This process, some believe, came about as a means for animals
inhabiting freshwater environments to regulate the composition of their
body fluids during a constant influx of water. Essentially, the GFR in fish
depends primarily on its hydration state and/or the availability of
freshwater in the environment in which it lives (Braun and Dantzler,
1997). Thus, the more freshwater that is available to the organism, the
more filtration occurs across the glomerulus, allowing for the elimination
of excess water and metabolic wastes. In times, when water is less available,
there is a tendency to conserve body water by not filtering it for once it is
filtered, water is potentially lost from the body (Braun and Dantzler, 1997).
In hagfish, the total filtering surface of the glomeruli is larger than in most
marine teleosts and as large as that of many elasmobranchs and freshwater
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Fish Osmoregulation
teleosts (Hickman and Trump, 1969). However, their low dorsal aortic
blood pressure and reduced water influx due to their osmoconformity is
reflected in their low rates of glomerular filtration, averaging about 0.25 ml
kg1 h1 in Eptatretus stouti and Myxine glutinosa, and similar to rates
measured in marine teleosts (Johansen, 1960; Hickman and Trump, 1969;
Riegel, 1978, 1986). In contrast, the GFRs of freshwater lamprey,
averaging 28 ml kg1 h1, are almost 100 times higher than its fellow
agnathan and are 5-7 times higher than that in freshwater teleosts
(Moriarty et al., 1978; Evans, 1979). In half-strength seawater, the GFR of
lampreys is 80% lower than that in freshwater (McVicar and Rankin,
1985; Evans, 1993).
The rates of glomerular filtration in marine elasmobranchs average
about 3.5 ml kg1 h1, which are much higher than marine teleosts and
approach those of freshwater teleosts (Hickman and Trump, 1969; Evans,
1979, 1993). Their open glomeruli, with a very large filtering surface,
suggest that even these observed rates of filtration are lower than the
potentially maximal rates (Hickman and Trump, 1969). As one might
expect, upon exposure to more dilute conditions, the GFRs of euryhaline
elasmobranchs increase (Evans, 1993). Glomerular filtration rates in
freshwater Potamotrygonidae average approximately 8.3 ml kg1 h1,
significantly higher than marine elasmobranchs and euryhaline
elasmobranchs in dilute conditions (Goldstein and Forster, 1971).
One of the major differences between the freshwater and marine
kidney is the role of glomerular filtration in urine formation. In freshwater
teleosts, the rate of glomerular filtration is high (~ 4 ml kg1 h1),
contributing to the excretion of a dilute, hypoosmotic urine (Nishimura
and Imai, 1982). In a marine environment, water is at a premium and a
glomerulus, which aids in the elimination of body water, essentially
becomes a liability. Thus, GFR in these fish is low (~ 0.5 ml kg1 h1) and
various stages of glomerular degeneration is observed, the end result being
fish that are completely aglomerular (Lahlou et al., 1969; Nishimura and
Imai, 1982; Beyenbach, 1986; Baustian et al., 1997).
a. Glomerular intermittency
When euryhaline fishbe it agnathans, elasmobranchs or teleostsare
transferred from one salinity to another, the change is reflected in the rate
of glomerular filtration. There are two important ways that body water can
be regulated through GFR; by changes in the amount of blood flowing into
315
the glomerulus (the renal perfusion pressure), or by changing the number

of glomeruli that are filtering (glomerular intermittency). Partly as a
consequence of differences in GFR, the urine flow rates in marine teleosts
with glomerular (0.03 to 0.89 ml kg1 h1) or aglomerular kidneys (0.03 to
0.45 ml kg1 h1) are greatly reduced as compared to those of freshwater
fish (4 ml kg1 h1) (Hickman and Trump, 1969). What may be apparent
is that despite rather large differences in the urine flow rates of freshwater
versus marine teleosts, there is no substantial difference between
glomerular and aglomerular marine fish. This is due in part to glomerular
intermittency, where the number of operational glomeruli changes
depending on environmental conditions and results in variations in GFR.
Glomerular intermittency has been well described in hagfish, the river
lamprey, Lampetra fluviatilis, the lesser spotted dogfish shark, Scyliorhinus
canicula as well as the rainbow trout, Oncorhynchus mykiss (Brown, 1980;
Brown and Green, 1987). In all species, three functional glomerular types
have been found; glomeruli that are perfused with blood and filtering,
those that are non-perfused and non-filtering and those that are perfused
but non-filtering. Changes observed in GFRs are essentially reflections of
alterations in filtration by single nephrons (single nephron glomerular
filtration rate; SNGFR) which can occur in at least two ways. First, the
same number of glomeruli filter but all are filtering at a different rate.
Second, a different number of glomeruli are filtering, i.e., some of the
glomeruli could stop filtering entirely while others continue to filter at a
relatively normal rate. The end result in both cases is the same but the
mechanism by which it is accomplished is different.
In fish, regulating the number of filtering glomeruli appears to be the
most important mechanism for changing the rate at which the whole
kidney filters. An exception is the river lamprey (Lampetra fluviatilis).
Under freshwater conditions, almost all the glomeruli are filtering but
when exposed to brackish or seawater conditions, decreases in GFR are
due to a reduction in the filtration rate of all the nephrons, likely due to
changes in the blood pressure of the renal arteries (Moriarty et al., 1978;
Logan et al., 1980a; Rankin et al., 1980; McVicar and Rankin, 1985; Brown
and Rankin, 1999). Since lamprey do not have a renal portal system,
which would allow the continuation of tubular function when glomerular
blood flow is reduced, it is believed that they keep all glomeruli perfused
because this is the only way blood gets to the nephron tubule. Similar to
freshwater lamprey, almost all of the glomeruli are filtering in dogfish,
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Fish Osmoregulation
despite being in a marine environment (Brown and Green, 1987).

However, one must keep in mind that the osmoregulatory strategy of
elasmobranchs results in an osmotic influx of water, making their rates of
glomerular filtration and urinary excretion more comparable to freshwater
fish. Infusion of adrenaline into dogfish reduces the number of filtering
glomeruli from 94 to 70%, in contrast to observations in lamprey in
response to seawater in which the number of filtering glomeruli never
changed. Interestingly, adrenaline also increased SNGFR in dogfish,
resulting in an overall diuresis (Brown and Green, 1987).
In contrast to both lamprey and dogfish, only about 45% of the
glomeruli are perfused and actively filtering in rainbow trout under
freshwater conditions and approximately 5% of glomeruli are filtering in
the kidneys of seawater-adapted trout (Brown et al., 1978, 1980; Amer and
Brown, 1995). Interestingly, there appears to be a large glomerular reserve
in both freshwater and marine, there being a large proportion of perfused
but non-filtering glomeruli in trout found in either environment. The
bottom line is that over 90% of the glomeruli are out of commission in
seawater, essentially rendering a glomerular trout aglomerular in seawater
and explaining why urine flow rates are similar in marine teleosts
regardless of the presence of glomeruli. Another example of this is the
Prussian carp, Carassius auratius gibelio, that decreases the number of
perfused glomeruli upon exposure to seawater by half and > 90% of its
glomeruli disappear over the next three months in seawater (Elger and
Hentschel, 1981; Elger et al., 1984, reviewed by Beyenbach, 1986). Since
Mg2+, SO 42, Na+ and Cl are the major urine electrolytes and osmolytes
irrespective of glomeruli, urine formation is highly dependent on tubular
activities in both glomerular and aglomerular fishes (see below; reviewed
by Beyenbach, 2004).
ii. Tubular modification: Reabsorption and secretion
In most fish, the renal filtrate passes through the glomerulus and is then
further modified by the renal tubule system, making tubular modification
the second step in renal osmoregulation. Of course, this is not the case in
aglomerular fish, in which urine formation starts at the level of the renal
tubule, making the urine flow rate significantly greater than their nonexistent GFR. However, for the most part, urine flow rates are significantly
less than GFRs due to tubular water reabsorption that is driven by the
reabsorption of ions. The only glomerular exception here is hagfish, in
317
which urine flow rates are almost equal to GFRs, indicating little tubular
reabsorption of water as might be expected when in a nearly isoosmotic
solution (Evans, 1979). In freshwater lamprey, urine flows are
approximately 50% of GFR, indicating that water is reabsorbed by the
kidney tubule (Moriarty et al., 1978; Logan et al., 1980a). When in half
strength seawater, lampreys show a decrease in GFR and an increase in the
tubular reabsorption of water resulting in an 80% reduction in urine flow
rate (Rankin et al., 1980; McVicar and Rankin, 1985). In marine
elasmobranchs, 70-85% of the filtered water is reabsorbed by the tubules.
Urine flows increase when euryhaline elasmobranchs are exposed to more
dilute conditions, in part due to a decrease in tubular reabsorption (Evans,
1993). In freshwater teleosts, only 45% of the filtered water is reabsorbed
by the kidney tubules (reviewed by Wood and Patrick, 1994). However,
tubular reabsorption increases to approximately 75% in marine teleosts.
In most vertebrates, the renal tubules normally reabsorb between 35%
and 97% of the filtered Na+ and Cl, the highest fractional reabsorption
being found in some freshwater species that have little access to these ions
(reviewed by Braun and Dantzler, 1997; Dantzler, 2003). The fractional
reabsorption in marine and possibly freshwater elasmobranchs appears to
be even lower and in the primitive marine hagfishes, which conform
osmotically to the surrounding seawater. There is little to no reabsorption
of filtered sodium along the archinephric ducts (Stolte and SchmidtNielsen, 1978; Evans, 1979; Dantzler, 2003). However, it should be kept
in mind that most data on fractional reabsorption for the entire kidney
come from clearance studies that give only the net difference between the
amount filtered and amount excreted in the ureteral urine (Braun and
Dantzler, 1997). Within the different parts of the tubule system there is
both net reabsorption and net secretion of Na+, Cl as well as other
solutes. The extent to which water and ions are reabsorbed or secreted is
variable amongst fishes and is highly dependent on the osmolality of the
environment and kidney morphology, with each segment of the kidney
tubule contributing differently to salt and water balance. In the following
paragraphs, the transport properties and the osmoregulatory relevance of
each kidney tubule segment will be described in detail.
a. Proximal tubule I
The primary urine leaves the glomerulus and in most fish enters the first
segment of the proximal tubule (proximal tubule I). In tetrapods and birds,
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Fish Osmoregulation
the proximal tubule is a major site of Na+, Cl and Mg2+ reabsorption. Due
to their osmoconformity, there is some debate on whether the archinephric
ducts of hagfish, which are morphologically similar to the proximal tubule
of tetrapods, reabsorb a small amount of Na+ (Evans, 1979; Braun and
Dantzler, 1997). However, there is clear evidence for net Na+ reabsorption
along the proximal tubule of the freshwater lamprey, albeit minor (Logan
et al., 1980a). Micropuncture analysis of samples taken from various points
along the lamprey proximal segment indicate that no more than 10% of
filtered water is reabsorbed by these segments and fluid within the
proximal tubule remains isoosmotic to blood (Logan et al., 1980a). Thus,
the ion transport by the proximal tubule in freshwater lampreys is
insignificant when compared to other segments (see below), consistent
with evidence indicating that Na+, K+-ATPase activity in the lamprey
proximal segment is much lower than in the distal segment (Natochin,
1972; Logan et al., 1980a). Upon acclimation to 50% seawater, tubular
water reabsorption in lamprey increases by 40% compared to freshwater
conditions contributing to a dramatic reduction in urine flow rates
(Rankin et al., 1980). But, similar to the freshwater condition, no more
than 9% of the filtered water was reabsorbed by the proximal segment,
suggesting that the increased tubular water reabsorption likely occurs
further down the tubule (Logan et al., 1980b). Rankin and coworkers
(1980) suggested that the main function of the proximal segment in
seawater lampreys is to secrete Mg2+ and SO42, however, this is yet to be
determined conclusively.
Based on the tubular fluid to plasma ratios, there appeared to be some
minor reabsorption in proximal tubule I of the little skate, Raja erinacea
(Stolte et al., 1977). However, no clear quantitative information is
available concerning the magnitude of proximal tubule I NaCl
reabsorption in elasmobranchs or teleosts. If proximal tubule I has even an
average water permeability, then some NaCl reabsorption in this tubule
segment may occur in marine glomerular fish to take advantage of the fact
that the reabsorption of solutes would be accompanied by an osmotically
equivalent quantity of water. In this way, proximal tubule I NaCl
reabsorption would allow a marine fish to recover filtered fluids, the excess
NaCl could then be secreted further down the nephron (see below). One
must keep in mind, however, that in glomerular marine teleosts,
glomerular filtration is reduced dramatically. Therefore, little NaCl
reabsorption is needed to recover fluids lost by filtration. In theory, very
319
little NaCl reabsorption should occur across proximal tubule I in

freshwater glomerular fish, since nearly all (96%) the filtered NaCl is
eventually reabsorbed but only 47% of filtered water is recovered
(Beyenbach, 2000). In freshwater fish, most of the NaCl would have to be
actively reabsorbed across more water impermeable regions of the nephron
(i.e., the distal tubule) or by the urinary bladder.
Based on the observation that marine aglomerular teleosts lack
proximal tubule I, Hickman and Trump (1969) suggested that proximal
tubule I in freshwater teleosts was likely to be responsible for the
reabsorption of valuable solutes that had entered the kidney tubule by
filtration, i.e., glucose, amino acids and macromolecules as well minerals
such as Mg2+ and Ca2+ (Bijvelds et al., 1998; Beyenbach, 2000). Since
aglomerular teleosts lack the ability to filter these substances, they have no
need to reabsorb them, explaining their proximal tubule I deficiency.
Interestingly, 80% of the filtered glucose in hagfish is reabsorbed across
their proximal tubule I-like archinephric ducts (Munz and McFarland,
1964; Hickman and Trump, 1969), suggesting that marine glomerular fish
may also use proximal tubule I for the recovery of valuable solutes.
Furthermore, the structural similarity of lamprey proximal cells to those of
the proximal segments of glomerular teleosts and higher vertebrates
suggests that its main function is also the reabsorption of filtered
macromolecules (Logan et al., 1980a). However, this theory was called into
question recently when evidence for net tubular secretion and subsequent
phlorizin-sensitive reabsorption of glucose in the aglomerular, Lophius
americanus, a fish believed to be lacking proximal tubule I, had surfaced
(Malvin et al., 1965; Braun and Dantzler, 1997). This was contradicted by
a study using kidney brush border membrane vesicles made from the
aglomerular toadfish, Opsanus tau that provided no evidence of a
phlorizin-sensitive glucose reabsorptive system (Wolff et al., 1987).
However, it is not known whether toadfish secrete glucose to be
reabsorbed, as is believed to be the case for goosefish.
Regardless, proximal tubule I likely does not play a major role in net
NaCl balance in freshwater teleosts nor in hagfish, which are practically
isoosmotic to their environment. However, this tubule segment
contributes to NaCl and water balance to a certain extent in lamprey,
which are the only fish, to date, to clearly exhibit net reabsorption across
the proximal tubule in its entirety, as well as marine elasmobranchs and
glomerular marine teleosts. Detailed models on the cellular transepithelial
320
Fish Osmoregulation
NaCl reabsorptive processes in proximal tubule I are available only for

amphibians. However, it is believed that the fractional reabsorption of Cl
is essentially the same as that of Na+ along proximal tubule I of lampreys,
elasmobranchs and teleosts based on micropuncture or microperfusion
studies (Braun and Dantzler, 1997). In addition, lumen-negative
transepithelial potential and net transepithelial Na+ reabsorption occurs
against an electrochemical gradient in the proximal tubule of freshwater
adapted teleosts, suggesting active Na+ transport and passive Cl transport
(Braun and Dantzler, 1997).
b. Proximal tubule II
The second segment of the proximal tubule (proximal tubule II) appears
to be responsible for the secretion of many different osmolytes in both
elasmobranchs and marine teleosts (Beyenbach and Frmter, 1985;
Sawyer and Beyenbach, 1985; Beyenbach et al., 1986; Cliff et al., 1986;
Cliff and Beyenbach, 1992; Evans, 1993). Aside from the collecting tubule
and duct, proximal tubule II is the only tubular segment found in
aglomerular marine teleosts. In contrast, neither hagfish nor lamprey are
believed to possess this segment (Hickman and Trump, 1969; Hentschel
and Elger, 1989; Evans, 1993).
Overall, most fish show a net secretion of divalent ions and both Na+
and Cl along the entire length of the proximal tubule (not distinguishing
between proximal tubule I and II) and there is even evidence of net
secretion in freshwater (Hickman and Trump, 1969; Braun and Dantzler,
1997; reviewed by Dantzler, 2003). The first direct observation of NaCl
and fluid secretion in proximal tubules occurred serendipitously. In his
1986 review, Klaus Beyenbach explains how he made his discovery while
using an isolated tubule approach to study Mg2+ secretion in the proximal
tubules of the glomerular winter flounder, Pseudopleuronectes americanus
(Beyenbach, 1982, 1986). This approach requires the isolated tubule to be
filled with oil in order to collect the luminal contents for analysis. No
sooner had the tubule lumen been filled with oil than the oil column broke
up at several places along the length of the tubule as a consequence of fluid
being secreted into the tubule. Over time, the fluid spaces between oil
droplets grew and typically expelled all the oil from the tubule lumen
within an hour. Beyenbach (1982) went on to discover that the dominant
electrolytes in the secreted fluid were surprisingly Na+ and Cl, in nearly
the same concentration as the peritubular bath. The tubule segments also
321
secreted Mg2+ and SO 42 and the secretion of these divalent ions was
found to increase the total rate of fluid secretion. However, the basic
mechanisms of fluid excretion appeared to be driven by the secretion of
NaCl, as fluid secretion continued, albeit at lower rates, with the removal
of MgSO4 but was inhibited by the replacement of Na+ or Cl with large
organic ions.
Like the flounder, the proximal tubule II of the dogfish shark, Squalus
acanthias, secretes predominantly NaCl in concentrations not significantly
different from those in the bath. The secreted fluid becomes slightly
hyperosmotic to the bath due to the secretion of significant concentrations
of Mg2+ and SO 42 (Sawyer and Beyenbach, 1985). Significant Mg2+,
PO 42 and SO 42 secretion, as indicated by high proximal tubule fluid to
plasma ratios, has also been shown to occur in proximal tubule II of the
little skate, Raja erinacea (Stolte et al., 1977). Evidence indicates that the
mechanism of NaCl secretion in dogfish proximal tubule II includes an
apical, cAMP sensitive Cl channel (Beyenbach and Frmter, 1985). On
the basolateral side of the tubule, the intracellular voltage was sensitive to
ouabain (an inhibitor of Na+, K+ ATPase) and furosemide (an inhibitor
of Na+, Cl co-transport). The effects of cAMP, furosemide and ouabain
on NaCl secretion and tubule electrophysiology are consistent with
secondary active transport of NaCl, where the basolateral membrane Na+,
K+ ATPase generates the primary driving force. In brief, the generation of
low intracellular Na+ concentrations via the Na+, K+ ATPase provides a
large electrochemical gradient for Na+ entry from the peritubular bath.
The energy of this Na+ gradient is coupled to the entry of Cl by way of
the furosemide sensitive Na+, Cl co-transporter. Na+ arriving in the cell
is returned to the bath via the Na+, K+ ATPase, but Cl diffuses out of the
cell through apical membrane Cl channels, the conductivity of which is
regulated by cAMP. The cellular secretion of Cl into the tubule lumen
appears to be electrically balanced by the paracellular movement of Na+
from bath to lumen. There are slight differences in NaCl secretion in
teleosts but for the most part the mechanism remains the same.
Net NaCl and water secretion has also been observed in the killifish,
Fundulus heteroclitus, a glomerular, euryhaline teleost (Beyenbach and
Frmter, 1985; Cliff and Beyenbach, 1992). Net secretion of NaCl and
water may be particularly important in euryhaline teleosts for the
maintenance of renal excretion during periods of glomerular intermittency
when the number of filtering glomeruli is reduced during adaptation to
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Fish Osmoregulation
seawater (Beyenbach and Frmter, 1985). Along the same lines, tubular
secretion is important in producing urine in aglomerular fish. In both
cases, the cessation or lack of glomerular filtration must be compensated
by tubular changes; in this case, primary urine formation depends mainly
on tubular secretion, at the same time tubular reabsorption is reduced
(Beyenbach and Frmter, 1985). Here the tubular secretion of NaCl could
serve to excrete excess Na+ and Cl, which seawater fish must do, and to
excrete organic solutes via cotransport with Na+ or Cl (Beyenbach and
Frmter, 1985). It is not yet known whether net secretion along the
proximal tubule II changes to net reabsorption when euryhaline teleosts
are adapted to freshwater. However, preliminary data on killifish adapted
to freshwater suggest that some, if not all, nephrons still secrete Na+ and
Cl (Cliff and Beyenbach, 1988).
In theory, the secretion of Na+, Cl, Mg2+ and SO 42 along proximal
tubule II without further modification could lead to a final urine osmolality
that exceeds that of plasma. In fact, data reported for the killifish, Fundulus
kansae indicates that a urine hyperosmotic to plasma is possible in fish
(Stanley and Fleming, 1964; Fleming and Stanley, 1965). Furthermore, a
recent study by McDonald and Grosell (2006) on the aglomerular gulf
toadfish, Opsanus beta, measured urine osmolalities that were at times
greater than those in plasma at a range of environmental salinities.
c. Nephron loop
Only lamprey and elasmobranchs have a loop within their nephron
structure. In lamprey, the proximal and distal tubule themselves are
arranged in a loop and in freshwater, the loop arrangement itself does not
appear to play a role in the concentration of urine or the tubular handling
of particular solutes. However, when in marine conditions, lamprey have
the ability to produce a urine that is hyperosmotic to plasma and it is
believed that the lamprey nephron loop may contribute to its
concentrating ability under these conditions (Youson and McMillan,
1971; Natochin, 1977; Logan et al., 1980b).
In comparison, the nephron structure in the elasmobranch is more
complicated than that of the lamprey. Instead of just one nephron loop,
the elasmobranch kidney has five loops arranged in countercurrent
fashion, weaving in and out of the sinus zone and the enclosed bundle zone
(Lacy and Reale, 1986; Friedman and Hebert, 1990). This countercurrent
system is unique to marine and euryhaline elasmobranchs and is absent in
323
the freshwater stingrays of the family Potamotrygonidae. Interestingly,

freshwater elasmobranchs have also lost the ability to retain urea,
suggesting that the nephron loops in the marine elasmobranch kidney may
be involved in recovering the 90-96% of the urea present in the glomerular
filtrate (Forster, 1967; Goldstein and Forster, 1971; Payan et al., 1973).
Furthermore, the fluid entering the marine elasmobranch collecting duct
has been shown to have already reached its final low concentration with
respect to urea (Thurau and Acquisto, 1969; Deetjen et al., 1972; Stolte
et al., 1977).
In mammals, the countercurrent organization of nephron segments
and capillaries is involved in the recycling and concentration of urea in the
inner medulla (Hogg and Kokko, 1979). The energy for this process in
mammals is provided by NaCl absorption from the water-impermeable
thick ascending limb of Henle, also known as the diluting segment. In
elasmobranchs, there is also a direct correlation between reabsorption of
Na+ and urea (Schmidt-Nielsen et al., 1972). Furthermore, a nephron
segment having morphological homology with the mammalian TAL has
been localized to the bundle zone in elasmobranch kidney that shows a low
permeability to water and active NaCl transport (Hentschel and Elger,
1987; Friedman and Hebert, 1990). With respect to urea, the
elasmobranch countercurrent model of Boylan (1972) predicted a zone of
low urea concentration around the terminal segment (i.e., collecting duct)
of the elasmobranch nephron that allows for the passive reabsorption of
urea. This was elaborated by Hentschel and coworkers (1986), who
predicted that solute gradients would exist between the dorsal and ventral
kidney sections that would support a passive reabsorption of urea in the
dorsal region. However, there is no evidence of urea zonation between the
two major kidney sections as measured urea and water content within
these sections were identical (Hentschel et al., 1986; Morgan et al., 2003a).
Friedman and Hebert (1990) determined that the permeabilities of the
tubule segments running through the peritubular sheath to water, urea and
Na+ differ, a general requirement for countercurrent exchange (Friedman
and Hebert, 1990). It is now believed that the peritubular sheath
segregates the dorsal tubule bundle of a single nephron and creates a urea
gradient from the tip to the end of the bundle, forming a countercurrent
exchange microenvironment (Lacy et al., 1985; Friedman and Hebert,
1990; Hentschel et al., 1998). The complexity of the elasmobranch kidney
makes this hypothesis extremely difficult to test. However, consistent with
324
Fish Osmoregulation
a localized countercurrent exchange system within the dorsal bundle is the

presence of a facilitative diffusion urea transporter, skUT, discovered on
the brush border membrane on the dorsal bundle tubule segments
(Morgan et al., 2003b). Furthermore, both skUT expression and a Na+linked urea transport have been measured in the ventral kidney section,
suggesting a role for this section in urea reabsorption as well (Morgan et al.,
2003a, b). When adapting to environments of lower salinity, marine
elasmobranchs reduce their plasma urea concentrations. This regulatory
adjustment is accomplished largely by a decrease in renal urea reabsorption
(Goldstein et al., 1968; Goldstein and Forster, 1971; Forster et al., 1972;
Schmidt-Nielsen et al., 1972; Piermarini and Evans, 1998; Cooper and
Morris, 2004).
d. Distal tubule and collecting duct
Found in lamprey, elasmobranchs as well as freshwater and euryhaline
teleosts, the distal tubule enables the reabsorption of osmolytes without
the accompaniment of water, due to its low water permeability. In this way,
urine essentially becomes diluted as a consequence of salt recovery and the
excess water is trapped within the tubule and excreted. Because of its
function, the distal tubule is not found in stenohaline marine glomerular
and aglomerular teleosts as its presence would be detrimental due to their
need to retain water and eliminate excess salt. The distal tubule is also
absent in hagfish.
In freshwater lamprey, most of the NaCl reabsorption occurs between
the beginning of the distal tubulewhich is the ascending limb of the
lamprey nephron loopand the end of the collecting duct. Greater than
95% of the filtered NaCl is reabsorbed across both tubular segments but
mainly across the distal tubule, resulting in a significant dilution of the
tubular fluid, despite substantial water reabsorption (Logan et al., 1980a).
In fact, approximately 45% of the filtered water is reabsorbed by the
freshwater lamprey kidney, most of it within the distal and collecting
tubules. However, one must keep in mind that the lamprey GFR is 5-7
times higher than a freshwater teleost; correspondingly, tubular
reabsorption might be higher to compensate. In contrast, almost 90% of
the filtered water is reabsorbed by the lamprey kidney in the marine
environment and most of this is believed to occur in the collecting ducts
(Logan et al., 1980b). As a result, urine flow rate in marine lamprey is 10%
that in their freshwater counterparts.
325
There are few studies outlining the micropuncture analysis of

elasmobranch distal tubule fluid, specifically due to its complicated kidney
morphology. However, fluid from the end collecting duct of the little skate,
Raja erinacea, showed much higher concentrations of Mg2+, PO42+ and
SO 42, than that those measured in plasma (Stolte et al., 1977). In contrast,
Na+ and Cl concentrations were slightly lower than plasma
concentrations, indicating some reabsorption. While the urine remained
isoosmotic in the proximal tubule, along the collecting ducts and in the
final urine the osmolality of the tubular fluid was below plasma values
(Stolte et al., 1977). This dilution became more pronounced when fish
were kept in 75% seawater and was believed to be due to an increased
reabsorption of Na+ (Stolte et al., 1977).
In freshwater teleosts, more that 90% of the filtered NaCl is
reabsorbed across the distal tubule, which has little to no permeability to
water (Nishimura and Imai, 1982; reviewed by Braun and Dantzler, 1997;
Dantzler, 2003). In the absence of the distal tubule in stenohaline marine
teleosts, it appears that some reabsorption occurs in the collecting ducts
and urinary bladder (see below) (reviewed by Braun and Dantzler, 1997;
Dantzler, 2003). Microperfusion studies of the distal tubule segments
amongst vertebrates reveal a net Cl reabsorption and a lumen-positive
transepithelial potential, which are dependent on the presence of both
Na+ and Cl in the lumen (Dantzler, 2003). Both Cl reabsorption and the
lumen-positive TEP are reduced or eliminated by the addition of
furosemide or bumetanide to the lumen or ouabain to the peritubular fluid.
This supports Cl reabsorption occurring by a Na+-coupled mechanism,
the energy for which is derived from Na+, K+, ATPase at the basolateral
membrane that maintains a low intracellular sodium activity. The gradient
established by the Na+, K+, ATPase drives the coupled, electroneutral
entry of Na+, 2 Cl and K+ into the cell across the luminal membrane via
a furosemide or bumetanide sensitive carrier (NKCC). Cl then exits the
cell across the peritubular membrane down an electrochemical gradient
via a Cl conductive pathway and a coupled electroneutral carrier with
K+ .
The Role of the Urinary Bladder
There is very little research conducted on the role or even the existence
of the urinary bladder in agnathans or elasmobranchs. However, in most
teleosts, paired mesonephric ducts leave the kidney, unite and open into
326
Fish Osmoregulation
a widened duct commonly termed the urinary bladder (Hickman and

Trump, 1969; Nishimura and Imai, 1982). One or more sphincters guard
the outlet through the urogenital papilla, which enables the urine to be
stored in the bladder for some time prior to natural discharge (Curtis and
Wood, 1991). Freshwater rainbow trout appear to urinate intermittently in
bursts at 20-30 minute intervals, and natural rates of urine flow and NaCl
excretion are at least 20 and 40% lower, respectively, than rates
determined by internal catheters which bypasses bladder function (Curtis
and Wood, 1991). Thus, at least in freshwater teleosts, urine appears to be
stored in the bladder for some time prior to discharge and significant
reabsorption occurs during the period (Curtis and Wood, 1991). Like the
distal tubule, the urinary bladder of freshwater teleosts is relatively
impermeable to water as compared to the bladders of marine teleosts but
is much more permeable to Na+ and Cl (Hirano et al., 1973; Fossat and
Lahlou, 1977; Demarest, 1984). In general, its main function is to reduce
renal ion losses and contribute to water excretion.
In contrast, urinary bladder water permeability is variable among
seawater fishes but is generally higher than the permeability of the
freshwater bladder (Hirano et al., 1973). In the euryhaline flounder,
Platichthys stelatus, the rate of fluid reabsorption by bladders in seawateracclimated fish are double reabsorption rates in freshwater acclimated fish
(Demarest, 1984). While the urinary bladder performs a storage function
within some marine teleosts, for the most part its main function is the
reabsorption of water, participates significantly in the osmoregulation of
marine fish (Lahlou et al., 1969; Howe and Gutknecht, 1978; McDonald
et al., 2002; McDonald and Grosell, 2006). In the aglomerular gulf
toadfish, there is clear evidence that the reabsorption of Na+ across the
urinary bladder is dependent on water requirements, with reduced Na+
reabsorption across the bladder occurring in hyposaline environments
(Hirano et al., 1973; McDonald and Grosell, 2006). The reabsorption of
Na+ drives the reabsorption of water in the marine teleost bladder and its
low permeability to Mg2+ and SO 42 leaves these divalent ions to
concentrate. This selective permeability of the marine urinary bladder to
monovalent over divalent ions fueled the belief that tubule Na+
reabsorption was coupled to Mg2+ secretion in marine fishes (Beyenbach
and Kirschner, 1975; Howe and Gutknecht, 1978).
327
CONCLUSIONS
In freshwater lamprey, it is the distal tubule and collecting duct that are
most important in terms of osmoregulation, with the nephron loop playing
a larger role in the urine-concentrating ability in seawater. Aside from the
complex kidney morphology involved in the reabsorption of urea, urine
formation and osmoregulatory processes in marine elasmobranchs are
similar to teleost fish. These similarities become more apparent in the
freshwater stingray, Potamotrygonidae, whose kidney morphology and
function are very similar to freshwater teleosts. In freshwater teleosts, the
proximal tubule plays a minor role in osmoregulation whereas the
glomerulus, the distal tubule and urinary bladder play an important role in
the elimination of water and the recovery of osmolytes. In marine teleosts,
the proximal tubule, mainly proximal tubule II, is important for urine
formation, namely the secretion of Na+, Cl, Mg2+ and SO 42, which
cooperates with the collecting duct and urinary bladder to recover Na+
and Cl and water, leaving the divalent ions. The kidney of euryhaline
teleosts must shift from water excretion and monovalent ion conservation
in freshwater to water conservation in seawater.
Acknowledgements
MDM is funded by an NSERC (Canada) postdoctoral fellowship.
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Fish Osmoregulation
+0)26-4
12
Intestinal Transport Processes in
Marine Fish Osmoregulation
Martin Grosell
OSMOREGULATION IN MARINE FISH

The gastrointestinal tract (GIT) was recognized early to play a vital role in
marine teleost osmoregulation by absorbing water (Smith, 1930).
The extracellular fluids in marine teleosts, lampreys and chondrichthyan
fishes are hypotonic (~ 300 mOsm) (Rankin et al., 2001; Marshall and
Grosell, 2005; Taylor and Grosell, 2006a) with respect to the surrounding
seawater (~ 1000 mOsm), which results in diffusive water loss and salt
gain. To offset the diffusive water loss, marine teleost fish, marine lampreys
and seawater-acclimated sturgeon drink seawater at rates of 2-5 ml
kg1 h1 (Rankin et al., 2001; Rodriguez et al., 2002; Marshall and Grosell,
2005), of which 60-85% is absorbed by the intestine (Smith, 1930;
Shehadeh and Gordon, 1969; Sleet and Weber, 1982; Wilson et al., 1996;
Authors address: Rosenstiel School of Marine and Atmospheric Sciences, Division of Marine
Biology and Fisheries, University of Miami, 4600 Rickenbacker Causeway, 33145 Miami,
Florida, USA.
E-mail: mgrosell@rsmas.miami.edu
334
Fish Osmoregulation
Grosell et al., 1999). Intestinal water absorption is ultimately linked to salt

absorption and is the focus of the present chapter. For detailed discussions
of integrative aspects of whole animal salt and water balance in marine
fish, including renal and branchial contributions, the reader is referred to
other chapters in the present book and also recent reviews (Evans et al.,
2005; Marshall and Grosell, 2005; McDonald, 2007).
The esophagus and the intestine of marine fish contribute in
functionally distinct ways to marine teleost osmoregulation. The
esophagus displays high rates of NaCl absorption and is largely
impermeable to water, resulting in much lower NaCl concentrations and
osmotic pressure in stomach fluids compared to the ingested seawater
(Smith, 1930; Hirano and Mayer-Gostan, 1976; Kirsch and Meister, 1982;
Parmelee and Renfro, 1983; Wilson et al., 1996). The selective absorption
of NaCl, leaving other ions and water behind, is the function of both
passive and active transport with the latter ultimately driven by basolateral
Na+K+-ATPase (Hirano and Mayer-Gostan, 1976; Parmelee and Renfro,
1983). The apical transport processes involved in esophageal NaCl
absorption need to be fully characterized (Parmelee and Renfro, 1983). In
contrast to the largely water-impermeable esophagus, the intestine displays
water absorption in the order of 2-6 ml cm1 h1 (Grosell and Jensen, 1999;
Grosell et al., 1999, 2001, 2005; Wilson et al., 2002).
INTEGRATIVE SALT AND WATER MASS BALANCE
Water
Water loss to the marine environment is inevitable through renal
excretion and non-renal loss, most of which is likely to occur across the gill
surface. This diffusive water loss is compensated for by ingestion of
seawater as first recognized by Smith more than three-quarters of a
century ago (Smith, 1930). The control of seawater ingestion is
complicated and beyond the scope of the present text and has been
discussed recently in detail in several excellent reviews (Takei, 2000;
Loretz, 2001; Ando et al., 2003). Typical drinking rates of marine fish are
around 2 ml kg1 h 1, although multiple studies reports higher rates
(Marshall and Grosell, 2005) which may, at least in some cases, be elevated
due to handling stress. As mentioned, 60-85% of the ingested seawater is
absorbed by the gastrointestinal tract (Smith, 1930; Shehadeh and
Gordon, 1969; Sleet and Weber, 1982; Wilson et al., 1996; Grosell et al.,
Martin Grosell
335
1999) almost exclusively in the intestine as the esophagus is largely waterimpermeable (Hirano and Mayer-Gostan, 1976; Parmelee and Renfro,
1983). The remaining water is voided in the form of rectal fluids with
chemical compositions much different than the ingested seawater due to
fractional ion absorption, water absorption and secretions by the
gastrointestinal tract (Wilson et al., 1996, 2002; Grosell et al., 2001, 2004).
Monovalent Ions
Water absorption is ultimately linked to salt absorption (Skadhauge, 1974;
Mackay and Lahlou, 1980; Ando et al., 1986; Usher et al., 1991), so it is
perhaps not surprising that 98, 96 and 75% of ingested Na+, Cl and K+,
respectively, is absorbed along the gastrointestinal tract (Fig. 12.1). While
the esophageal and intestinal absorption of salt is necessary for water
absorption, it presents an additional challenge for marine fish by adding to
the diffusive salt gain from the concentrated environment. The majority
of the Na+, Cl and K+ gained from the gastrointestinal tract is eliminated
across the gill surface with <1, 3 and 7%, respectively, excreted along with
the urine. For detailed discussions of renal function in marine teleosts and
branchial transport processes, the reader is referred to recent reviews and
chapters within this volume (Evans et al., 2005; Marshall and Grosell,
2005; McDonald, 2007).
Divalent Ions
The dominant electrolytes in marine teleost intestinal fluids are Mg2+ and
SO42 reaching concentrations in some cases > 100 mM (Smith, 1930;
Grosell et al., 2001, 2004; Taylor and Grosell, 2006a, b), which is well
above corresponding seawater levels of approximately 50 and 30 mM,
respectively. From Figure 12.1 it is evident that fractional absorption of
Mg 2+ and SO 42 is low (20 and 67 %, respectively) compared to Na+, Cl
and K+ despite substantial gradients from the high concentrations in the
intestinal lumen to the low extracellular fluid concentrations (< 1 mM).
Even with the relatively low absorption of Mg2+ and SO 42 marine fish
experience a net gain of these divalent electrolytes, mainly from the
intestine. Homeostasis is maintained despite this continuous gain by renal
excretion, as discussed elsewhere (McDonald, 2007). While it is clear that
SO 42 extrusion from intestinal epithelial cells (discussed below)
contributes to the limited SO 42 gain across the intestine, it is unknown as
336
Fish Osmoregulation
Fig. 12.1. Whole animal balance of water (ml kg1 h1) and main electrolytes (mol kg1 h1)
in a typical marine teleost fish. Positive values indicate net uptake while negative values
indicate loss and excretion. Mass balance of water is based on assumed rates of drinking,
voided rectal fluids and urine flow rates of 2.0, 0.4 and 0.3 ml kg1 h1, respectively.
Electrolytes ingested with drinking are calculated from typical seawater composition while
rectal loss of Na+, Cl, SO42 and K+ are calculated from average concentrations of these
ions in rectal fluids (see Fig. 6.1 in Marshall and Grosell, 2005) and assumed an intestinal
fractional fluid absorption of 80% (Grosell et al., 1999; Shehadeh and Gordon, 1969; Wilson
et al., 1996). The rectal loss of Mg2+ and Ca2+ is estimated from fractional Mg2+ loss and
absolute Ca2+ loss which includes ions voided as CO 2
3 precipitates (Wilson and Grosell,
2003). Rectal fluids contain high amounts of HCO3 and CO 32 in solution and considerable
quantities of CaCO3 precipitates formed within the intestine (Grosell et al., 1999; Wilson
et al., 1996; Wilson and Grosell, 2003). The value for rectal HCO 3/CO 2
3 excretion depicted
in this figure was chosen to yield charge balance in the rectal fluids but represents a
conservative estimate since it is lower than the three empirical values reported to date
(Wilson et al., 1996; Grosell et al., 1999; Wilson and Grosell, 2003).
Renal excretion rates of electrolytes are calculated from average concentrations of
these ions in marine teleost urine ( Hickman and Trump, 1969; Beyenbach, 2004) and a
urine flow rate of 0.3 ml kg1 h1. Net branchial rates of electrolyte exchange with the
environment were determined from the difference between net gastrointestinal
contributions and renal contributions to consist mainly of excretion of NaCl and acidic
equivalents. Note here that branchial transport rates may include transport across other
non-gastrointestinal and non-renal surfaces. The net excretion rate of H+ and NH 4+ was
chosen to yield charge balance at the gill and is well within reported excretion rates and has
recently been reported to reflect intestinal HCO 3/CO 32 excretion rates (Wilson and Grosell,
2003).
to how Mg2+ uptake is largely avoided despite a strongly favorable gradient

for uptake.
Intestinal fluid Ca2+ concentrations are substantially lower than
corresponding seawater levels (Smith, 1930; Grosell et al., 2001, 2004;
Taylor and Grosell, 2006a, b). The low Ca2+ concentrations in intestinal
Martin Grosell
337
fluids has led to numerous suggestions of substantial intestinal Ca2+

uptake in marine teleost (Hickman, 1968; Evans, 1993; Karnaky, 1998).
However, more recent investigations have revealed that low intestinal
fluid Ca2+ concentrations are the product of alkaline precipitation rather
than absorption (Wilson et al., 2002; Wilson and Grosell, 2003). High
luminal HCO 3 concentrations and alkaline conditions in the intestinal
fluids facilitate CaCO3 formation, which is often evident as white mucuscoated structures within the intestinal lumen and holding tanks of marine
fish. These precipitates can account for 30-65% of the ingested Ca2+,
presumably explaining, at least, in part the relatively low intestinal
fractional Ca2+ absorption (20%; Fig. 12.1). Excess Ca2+ gained largely
from the intestine is eliminated by the kidney (Hickman, 1965).
Luminal Alkalinity
A highly alkaline intestinal lumen containing high concentrations of
HCO 3 and CO 32 is a feature unique to marine fish that ingest seawater.
This phenomenon was first reported some three-quarters of a century ago
(Smith, 1930), but only recently was its functional significance for
osmoregulation unraveled (Grosell et al., 1999, 2001; Wilson et al., 2002;
Wilson and Grosell, 2003; Marshall and Grosell, 2005; Grosell, 2006;
Grosell and Genz, 2006; Taylor and Grosell, 2006a, b). Considering the
drinking rates and seawater concentrations of HCO 3, it became apparent
that the source of HCO 3 and CO 32 in the intestinal fluids was the
intestinal epithelium itself (Walsh et al., 1991) and that the rectal fluid
represents a substantial base secretion (Wilson et al., 1996; Wilson and
Grosell, 2003). Although intestinal base secretion is not directly involved
in the dynamic regulation of acid-base balance, it contributes significantly
to the entire animal acid-base exchange with the environment. However,
variations in intestinal base secretion appear to be perfectly compensated
for by adjustments in branchial acid excretion (Wilson and Grosell, 2003).
Intestinal Fluid Composition
The differential absorption of Na+, Cl, K+ and water and secretion of
HCO3 result in a unique chemical composition of fluids residing in the
intestinal lumen of teleost fish. Marine teleost intestinal fluids are typically
high in Mg2+ and SO 42, often 3-4 fold higher than corresponding
concentrations in seawater, due to Na+, Cl and water absorption (Wilson
et al., 1996, 2002; Wilson, 1999; Bury et al., 2001; Grosell et al., 2001,
338
Fish Osmoregulation
2004, 2005; Grosell and Wood, 2001; Grosell, 2006). In contrast, Na+, Cl
and K+ concentrations in intestinal fluids are considerably lower than in
seawater as a result of the active absorption of these ions. Luminal Ca2+
concentrations are also consistently lower than in seawater but this is due
mainly to CaCO3 precipitate formation within the intestinal lumen rather
than Ca2+ absorption by the intestine (Wilson et al., 2002; Wilson and
Grosell, 2003).
Salinity
The data in Table 12.1 represents the first report of detailed intestinal fluid
chemistry in freshwater-acclimated euryhaline teleosts (Tilapia aureus) and
show a comparison to seawater-acclimated individuals. The most
substantial differences in chemical composition between freshwater and
seawater acclimated fish are Mg2+ and SO 42 concentrations, which are
both very low in freshwater- and high in seawater-acclimated individuals.
The high luminal concentrations of these divalent ions are related to
drinking and differential absorption in seawater acclimated fish. Despite
seawater ingestion, seawater-acclimated tilapia have similar or lower Na+,
Cl and K+ concentrations in intestinal fluids compared to freshwateracclimated fish, which demonstrates the substantial absorption of Na+ and
Cl in particular by the intestine of seawater-acclimated fish. Another
marked difference between intestinal fluids is evident from the total CO2
concentrations that are greatly elevated in seawater-acclimated compared
to freshwater-acclimated fish. This difference is in agreement with earlier
reports from euryhaline fish acclimated to different salinities (Wilson,
1999) and reflects intestinal Cl/HCO 3 exchange in seawater acclimated
fish. Alkaline intestinal fluids associated with the high total CO2
concentrations in seawater acclimated fish very likely explain the
relatively low Ca2+ concentrations in seawater-acclimated tilapia. As
discussed above, alkaline precipitation of CaCO3 accounts for the majority
of Ca2+ ingested with seawater and is released from the gastrointestinal
tract with the rectal fluids as clearly visible white pellets (Walsh et al.,
1991; Wilson et al., 2002; Wilson and Grosell, 2003).
A more detailed study of the influence of salinity on intestinal fluid
composition in the marine teleost Opsanus betaa fish that tolerates
salinity fluctuations wellrevealed that osmotic pressure in the intestinal
lumen remains relatively constant over a wide range of salinities (Fig. 12.2)
and that plasma osmolality and intestinal fluid osmolality are strongly
165.4
3.1
12.5
6.4
73.2
2.3
13.5
336.4
FW
5.2
0.9
1.3
2.1
9.0
1.2
2.9
6.9
SW
101.2
154.2
4.5
4.4
112.7
139.3
81.3
504.6
Anterior
14.3
36.2
0.8
0.5
17.7
30.5
16.8
31.6
131.8
0.9
8.1
1.2
87.1
1.2
36.8
321.1
FW
11.4
0.3
0.8
0.4
4.8
0.8
4.3
4.9
16.7
33.3
0.6
0.6
5.5
23.7
6.8
36.9
SW
103.4
155.3
3.6
3.5
81.2
105.4
97.4
497.6
Mid
129.6
1.0
9.4
2.1
98.8
0.6
20.4
325.1
4.8
0.3
1.0
0.6
6.9
0.4
3.9
8.7
FW
112.8
146.2
5.1
2.8
93.6
81.9
99.0
502.3
Posterior
10.0
37.5
0.4
0.7
7.0
12.7
13.4
36.7
SW
122.6
1.6
7.2
2.4
96.4
2.1
10.9
288.0
7.8
0.6
0.6
0.8
8.9
1.3
3.6
13.6
FW
73.4
179.8
8.5
4.3
74.8
121.3
110.5
469.3
Rectal
26.1
24.5
2.3
1.5
15.5
17.2
30.2
26.6
SW
Composition of fluids obtained from the anterior, mid and posterior segments of the intestine as well as from the rectum of seawater- and freshwater-acclimated Tilapia aureus
(plasma osmolality of 303.1 3.5 and 485.7 34.2, respectively). Values are means SEM (n=6-9) with concentrations expressed in mM and osmolality expressed in mOsm.
Adult tilapia were maintained in 500 L flow through tanks in Virginia Key dechlorinated tap water at 22C and were fed commercial tilapia food pellets daily. Following gradual
acclimation to increasing salinity over 7 days, fish were maintained in flow-through natural seawater (22C) for 2 weeks prior to sampling. Food was withheld from both freshwater
and seawater tilapia 48-h prior to sampling of intestinal fluids. Intestinal fluid samples were obtained and analyzed as described in detail elsewhere (McDonald and Grosell, 2005).
Na
Mg2+
K+
Ca2+
Cl
SO42
TCO2
Osmolality
Table 12.1
Martin Grosell
339
340
Fish Osmoregulation
Fig. 12.2 Osmolality (mOsm), Na+ and Mg2+ concentrations (Mean SEM) in fluids
obtained from the posterior intestine in the gulf toadfish (Opsanus beta) at salinities ranging
from 5-70 ppt. Note multiple x-axes showing ambient osmolality and Mg2+ concentrations
(top) and salinity and Na+ concentrations (bottom). Data taken from a recent study of Gulf
toadfish salinity tolerance (McDonald and Grosell, 2005).
correlated and similar (McDonald and Grosell, 2005). Even though

plasma osmolality in seawater-acclimated tilapia was elevated compared to
freshwater individuals, this correlation between intestinal fluid and plasma
osmolality was also observed for tilapia (Table 12.1). The data presented
in Figure 12.2 demonstrate that at salinities above 15 ppt, the intestinal
fluid Mg2+ concentrations increase gradually with salinity reflecting
increased drinking rates and water absorption by the intestine. In contrast,
Na+ concentrations gradually decrease at salinities above 15 ppt despite
increasing ambient Na+ concentrations and drinking rates. The
remarkable ability of the intestine to absorb Na+ (and Cl, data not
shown) is illustrated by luminal concentrations of less than 20 mM in fish
exposed to salinities of 50 ppt (equivalent to 650 mM Na+).
Martin Grosell
341
Intestinal Transport ProcessesNaCl Absorption

The intestinal epithelium provides for osmoregulation by absorbing water.
The mechanisms of water transport across leaky epithelia is a highly
controversial topic (Larsen et al., 2002; Spring, 2002), but regardless of the
mechanism, water transport is tightly linked to the active absorption of Cl
and Na+ (Skadhauge, 1974; Mackay and Lahlou, 1980; Ando et al., 1986;
Usher et al., 1991).
Na+K+-ATPase and Na+:Cl Co-transporters
The active absorption of both Cl and Na+ is ultimately fueled by the
basolateral Na+ K+-ATPase (NKA) which extrudes 2K+ in exchange for
3Na+ (Skou, 1990; Skou and Esmann, 1992). The activity of this enzyme,
which is highly abundant in the lateral membranes of the marine teleost
intestine (Fig. 12.3), maintains low intracellular Na+ concentrations and
sustains a highly cytosol-negative membrane potential. Increased NKA
mRNA expression and enzymatic activity in the intestine of euryhaline
fish following transfer to seawater illustrate the importance of this enzyme
for successful seawater osmoregulation (Mackay and Janicki, 1978; Jensen
et al., 1998; Seidelin et al., 2000; Cutler and Cramb, 2002b).
The electrochemical Na+ gradient energizes not only transport of Na+
but also Cl and K+ across the apical membrane by the Na+:Cl (NC) and
Na+:K+:2Cl (NKCC) co-transport systems (Field et al., 1978; Frizzell
et al., 1979; Musch et al., 1982; Halm et al., 1985b) (Fig. 12.4) and has
recently been demonstrated to fuel HCO3 secretion and Cl absorption by
apical anion exchange (see below)(Grosell and Genz, 2006). Consistent
with co-transporter activity, uptake of Na+ is abolished in the absence of
luminal Cl while some Cl absorption persists even in the absence of Na+
in the mucosal fluids (Mackay and Lahlou, 1980; Grosell et al., 2001)
demonstrating additional Cl uptake pathways. As is the case for NKA,
NKCC mRNA expression in the intestinal epithelium of euryhaline
teleosts increases following transfer from freshwater to seawater (Cutler
and Cramb, 2002b), illustrating the importance of NKCC for marine
osmoregulation.
All studies reporting simultaneous measurements of net Na+ and Cl
fluxes show higher net absorption rates for Cl than for Na+ in vivo, in situ
and in vitro (Hickman, 1968; Smith et al., 1975; Field et al., 1980; Mackay
and Lahlou, 1980; Gibson et al., 1987; Musch et al., 1990; Marvao et al.,
342
Fish Osmoregulation
Fig. 12.3 Section of rainbow trout (65% SW) pyloric cecae immunolabelled for NKA with
alpha-5 monoclonal antibody (red) (Perry, SF, Grosell, M and Gilmour, KM, unpublished).
Sections were counterstained with DAPI nuclear stain (blue). Note the columnar cells with
basal nucleus typical of absorbing epithelia and the clear lateral staining for NKA. Objective
40.
1994; Loretz, 1996; Grosell and Jensen, 1999; Grosell et al., 1999, 2001,
2005), as reviewed in detail by Grosell (Grosell, 2006), which may be
explained partly by the stoichiometry of NKCC. However, since K+
concentrations are relatively low in seawater and intestinal fluids, NKCC
itself cannot explain the substantial excess net Cl absorption seen in many
cases.
Intestinal Anion Exchange and Cl Absorption
In addition to net Na+ and Cl absorption rates reported for eight species
in the thirteen studies listed above, five reports on a total of four species
of marine teleosts also documented net HCO3 secretion rates which agree
well with the difference between net Cl and Na+ absorption rates (Grosell
and Jensen, 1999; Grosell et al., 1999, 2001, 2005; Grosell, 2005, 2006).
These observations strongly suggest that Cl/HCO3 exchange contributes
significantly (10-71%; Grosell, 2006) to overall net Cl uptake in concert
with the Na+:Cl co-transporters discussed above. A consequence of the
Martin Grosell
343
Fig. 12.4 Schematic cellular model of transport processes in the intestinal epithelium of
marine teleost fish. Transcellular and/or paracellular fluid absorption is driven by active
NaCl transport fueled primarily by the basolateral Na+-K+-ATPase (~) which provides the
electrochemical Na+ gradient allowing for Na+, Cl and K + entry across the apical
membrane. Two parallel systems, the Na +,Cl and the Na+, K+, 2Cl co-transporters
account for Na+, K+ and a portion of Cl absorption, with the remaining Cl uptake occurring
via anion exchange (AE). The apical AE performs active transport of not only Cl but also
HCO3 resulting in high luminal HCO 3 concentrations and highly alkaline intestinal fluids.
Further, anion exchange is involved in Cl/SO 2
4 exchange and likely maintains the
substantial transepithelial SO 2
4 gradient (Pelis and Renfro, 2003). Endogenous metabolic
CO2 provides cellular HCO 3 via carbonic anhydrase for the apical anion exchange process
with the resulting H+ being extruded across the basolateral membrane via an NHE-like
transporter. The H+ extrusion across the basolateral membrane is critical for apical HCO3
secretion and ultimately relies on the activity of the basolateral Na+-K+-ATPase (Grosell and
Genz, 2006). A physical association of AE and carbonic anhydrase II (CAII) might explain
how local HCO3 concentrations on the luminal side of the apical membrane can reach
levels satisfying the thermodynamic conditions necessary for anion exchange (Grosell,
2006). Exchange of a metabolic waste product (CO2), which exerts limited osmotic
pressure, in exchange for an electrolyte provides an osmotic driving force for cellular water
uptake. Basolateral import of HCO 3 from extracellular fluids appears to contribute to
luminal HCO 3 secretion as well and may occur via Na+: HCO 3 co-transport (NBC). Based
on previous studies summarized by Grosell (2006), fluid absorbed by the intestinal
epithelium is hyperosmotic and highly acidic (Grosell et al., 2005; Grosell and Genz, 2006).
See text for further details.
344
Fish Osmoregulation
intestinal anion exchange is high concentrations of HCO 3 in intestinal

fluids of marine teleosts. While this was reported for the first time in the
first part of the previous century (Smith, 1930) and since has been
confirmed for a large number of marine teleosts (Cordier and Maurice,
1956; Shehadeh and Gordon, 1969; Walsh et al., 1991; Wilson, 1999;
Grosell et al., 2001; Wilson et al., 2002), it was not until recently that
attempts were made to understand the functional significance of intestinal
anion exchange. The first two attempts (Wilson et al., 1996; Wilson, 1999)
to implicate intestinal base secretion in dynamic acid-base balance
regulation were negative (Wilson et al., 2002) and a role for intestinal
anion exchange in osmoregulation became evident from in situ perfusion
studies of the lemon sole intestine (Grosell et al., 1999) and transport
studies on isolated intestinal segments from the Pacific sanddab and the
European flounder (Grosell et al., 2001, 2005).
The Cl uptake and HCO 3 excretion arising from the intestinal anion
exchange are the function of secondary active transport and appear to
occur via an apical anion exchange protein (Dixon and Loretz, 1986;
Ando and Subramanyam, 1990; Wilson et al., 1996; Grosell and Jensen,
1999; Grosell et al., 2001, 2005; Wilson et al., 2002). The cellular substrate
for apical anion exchange seems to be a mix of HCO 3 from extracellular
fluids imported across the basolateral membrane and cellular hydration of
endogenous CO2 from epithelial respiration. Studies to date suggest that
30-60% of the secreted HCO 3 is derived from cellular CO2 hydration
(Dixon and Loretz, 1986; Ando and Subramanyam, 1990; Grosell et al.,
2005; Grosell and Genz, 2006) but species-specific differences are quite
likely to exist.
Serosal HCO 3 appears to provide part of the substrate of apical anion
exchange (Ando and Subramanyam, 1990; Grosell et al., 2005; Grosell and
Genz, 2006) and observations of luminal HCO 3 secretions being
dependent on serosal Na+ and sensitive to serosal DIDS suggest the
involvement of a basolateral Na+: HCO 3 co-transporter (NBC). A
basolateral NBC may allow for HCO 3 import by the intestinal epithelium
and, thus, provide cellular substrate for the apical anion exchange. The
HCO 3 uptake across the basolateral membrane via NBC would be driven
by the favorable Na+ gradient established by the basolateral NKA (Fig.
12.4). It should be noted, however, that a role for basolateral NBC in
intestinal HCO 3 secretion remains to be conclusively demonstrated.
Martin Grosell
345
Cellular CO2 hydration is partly mediated by carbonic anhydrase

(Dixon and Loretz, 1986; Wilson et al., 1996; Grosell and Genz, 2006) and
yields not only HCO 3 but also H+, which must be excreted from the
intestinal cells in order to avoid reversal of the CO2 hydration. A close
association between the apical anion exchanger and carbonic anhydrase
has been recently suggested to facilitate Cl/ HCO 3 exchange by providing
high local HCO 3 concentrations (Grosell, 2006), as seen in mammalian
systems (Sterling et al., 2001a, b, 2002). Furthermore, since the intestinal
epithelium exhibits net base secretion, these H+ are extruded across the
basolateral membrane displaying polarization of HCO 3 and H+ excretion
from the intestinal epithelial cells (Grosell and Genz, 2006). Basolateral
H+ extrusion seems to occur via a Na+: H+ exchange mechanism (i.e.,
Gulf toadfish) and, thus, ultimately relies on the Na+ gradient established
by the activity of the basolateral NKA (Grosell and Genz,
2006) (Fig. 12.4).
Regardless of the mode of Cl entry across the apical membrane,
cellular Cl concentrations (Duffey, 1979; Smith et al., 1980) are above the
electrochemical Cl equilibrium and Cl readily leaves the epithelial cells
across the basolateral membrane via Cl channels (Loretz and Fourtner,
1988) or K+:Cl co-transporters (Halm et al., 1985a).
Anion exchange is also likely to be involved in sustaining the large
SO 42 gradient across the intestinal epithelium since active extrusion of
SO 42 occurs across the apical membrane in exchange for Cl in the winter
flounder (Pelis and Renfro, 2003).
Intestinal Transport ProcessesWater Absorption
While it is unequivocally clear that water absorption relies on net NaCl
uptake, the exact mechanism of water absorption remains unknown and
may involve both transcellular and paracellular pathways. Although water
channels (aquaporins) are present in the intestinal tissue, there is no direct
evidence that these proteins are involved in transepithelial water
movement (Alves et al., 1999; Cutler and Cramb, 2002a; Lignot et al.,
2002). However, several co-transport proteins, including K+:Cl,
Na+:glucose and NKCC transporters, are capable of water transport in
other vertebrate systems (Loo et al., 2002; Hamann et al., 2005) and may
contribute to transcellular movement across the marine teleost intestine.
346
Fish Osmoregulation
Composition of the Fluid Absorbed by the Intestine

Hypertonicity
A number of studies report simultaneous measurements of net Na+, Cl
and water absorption rates (Hickman, 1968; Shehadeh and Gordon, 1969;
Pickering and Morris, 1973; Skadhauge, 1974; Grosell and Jensen, 1999;
Grosell et al., 2001, 2005; Marshall et al., 2002) which allow the estimation
of the Na+ and Cl concentration in the water absorbed by the intestine.
The tonicity of the absorbed fluid has also be estimated from these
measurements (Grosell, 2006) to be hyperosmotic with respect to both the
intestinal fluids and the blood plasma. These estimates of osmotic pressure
yield an overall mean value of 647 mOsm, which takes into account that
Na+ concentrations in the absorbed fluids are substantially lower than
corresponding Cl concentrations and so additional cation(s) must be
absorbed along with the Na+ and Cl. The absorbed fluid osmolality
estimate is based on an assumed osmotic coefficient of 1 and the
conservative assumption that no other anions are transported across the
epithelium as discussed in detail elsewhere (Grosell, 2006).
The Missing CationAcidic Absorbate
From parallel and direct, although not simultaneous, measurements of
water transport and serosal acid secretion in the gulf toadfish intestine, the
H+ concentration in absorbed fluids was estimated to be 77 mM,
corresponding to a pH of 1.1 (Grosell and Genz, 2006). These findings
verify the earlier suggestions of a highly acidic intestinal absorbate in the
European flounder (Grosell et al., 2005) and demonstrate that in the gulf
toadfish, the majority of the missing cationic charge can be attributed to
H+ secreted across the basolateral membrane. Higher net absorption rates
of Cl rather than Na+ seem ubiquitous to intestinal fluid absorption by
marine teleosts (Grosell, 2006), suggesting that H+ secretion across the
basolateral membrane and thus acidic intestinal absorbate may be a
general feature common to this group.
Dual Role of the Intestine: Feeding vs Osmoregulation
The intestine of marine teleosts, in particular, serves for both digestion and
water absorption (as an important component of osmoregulation) but
interactions between these two processes have not been considered
Martin Grosell
347
extensively. Early reports demonstrated that intestinal fluid chemistry and,

very likely, osmoregulation was influenced by the presence of partly
digested food in the intestine (Smith, 1930). In a recent study, the
influence of the ingestion of two different natural food sources on the
chemical composition of intestinal fluid and the osmoregulatory status of
the marine gulf toadfish was examined (Taylor and Grosell, 2006b).
Feeding transiently alters the intestinal fluid chemistry, dramatically
decreasing Mg2+ and SO 42 concentrations immediately following feeding,
indicating reduced drinking and/or reduced fluid absorption. In support of
this interpretation are elevated Na+ concentrations in the anterior
segments of the intestine which also indicate limited water absorption
immediately following feeding (Taylor and Grosell, 2006b). In contrast,
Cl levels are greatly reduced in all segments of the intestine for up to
48 hours post-feeding. This decrease in intestinal fluid Cl concentrations
is seen despite of titration of the acidic stomach content (HCl) moving
into the intestine and is followed by highly elevated intestinal fluid HCO 3
concentrations. The latter suggests that intestinal anion exchange (see
above) is stimulated following feeding and that this process may play an
important role in reestablishing the alkaline intestinal fluids following
feeding (Taylor and Grosell, 2006b). Despite the obvious influence of
feeding on intestinal transport processes associated with osmoregulation,
only a minor disturbance of salt and water balance was evident from
plasma osmolality and ionic composition (Taylor and Grosell, 2006b).
Endocrine Control of Intestinal Salt and Water
Transport
Recently, an excellent review of the endocrinology of osmoregulation
(Takei and Loretz, 2005) distinguished between two classes of
osmoregulatory hormones, as defined earlier (Takei and Hirose, 2002).
The first class consists of fast, short-acting hormones exhibiting rapid
stimulation and transient secretion and includes angiotensin II, arginine
vasotocin (AVT), natriuretic peptides, vasointestinal peptide, urotensins
and perhaps guanylins. The second class of slow, long-acting hormones
includes cortisol, 1a-hydroxy-corticosterone, growth hormone (GH) and
prolactin (PRL), with the latter likely being implicated in freshwater
adaptation only. While the fast-acting hormones control the already
existing transport proteins, modulating activity in many cases via
phosphorylation/dephosphorylation, the slow and long-lasting hormones
348
Fish Osmoregulation
typically involve transcriptional modifications (see below) and de novo

synthesis of ion transporting proteins (Takei and Loretz, 2005).
The osmoregulatory role of the gastrointestinal tract in marine fishes
is intimately linked to the regulated drinking reflex which is controlled by
a diverse and complicated multitude of parameters. The present text is
limited to a discussion of the direct endocrine (and paracrine) regulation
of intestinal transport and the reader is referred to a number of recent
reviews for detailed descriptions of drinking rate regulation (Takei, 2000;
Takei and Tsuchida, 2000; Ando et al., 2003; Marshall and Grosell, 2005;
Takei and Loretz, 2005).
Only a subset of the candidates for the endocrine control of
osmoregulation in marine fish have been tested for potential action on the
salt and water transport across the intestine, leaving a great deal of
research yet to be done. The following paragraphs provide a brief review
of the most frequently studied endocrine reagents that act on intestinal
transport processes related to osmoregulation.
Guanylins
Guanylins form an intestinal peptide family which is involved in
controlling salt and water absorption in mammals (Takei and Loretz, 2005)
and their transcription appears to be upregulated upon seawater transfer
in European eel (Comrie et al., 2001a, b), suggesting a role in seawater
osmoregulation. However, the impact of this peptide family on intestinal
salt and water transport in marine teleosts awaits characterization.
Vasointestinal Peptide (VIP)
Vasoactive intestinal polypeptide (VIP) inhibits intestinal Na+ and water
transport in both freshwater- and seawater-acclimated tilapia (Takei and
Loretz, 2005) and appears to inhibit net Cl absorption both in eel (Uesaka
et al., 1995) and winter flounder intestine; in the latter case by increasing
the unidirectional Cl secretion (OGrady, 1989; OGrady and Wolters,
1990). The stimulation of luminal Cl secretion could be related to
activation of a CFTR-like Cl channel in the apical membrane (Marshall
et al., 2002).
Neuropeptide Y
Neuropeptide Y (NPY), isolated from the Japanese eel intestine potently
increases net salt absorption, as is evident from the increased serosa
Martin Grosell
349
negative transepithelial potential and the enhanced short circuit current.

NPY appears to act on the apical NKCC cotransporter since its effects can
be blocked by bumetanide (Uesaka et al., 1996).
Natriuretic Peptides
Atrial natriuretic peptide (ANP) is synthesized by the heart but also by
non-cardiac tissues including the intestine (Loretz et al., 1997). Further,
the presence of ANP-ergic neurons in the intestinal tissue indicates
paracrine intestinal action (Loretz, 1995). The effect of ANP on the
marine teleost intestine is inhibition of net salt absorption (measured by
short circuit current in Ussing chambers during radio-isotopic fluxes)
(OGrady et al., 1985; OGrady, 1989; Ando et al., 1992; Loretz, 1996;
Loretz and Takei, 1997) presumably by inhibiting NKCC. Release of ANP
in marine teleosts is stimulated by elevated plasma Na+ concentrations
and hypervolemia. It acts to correct Na+ concentrations and extracellular
fluid volume by reducing the intestinal salt and water absorption and by
stimulating branchial salt extrusion (Loretz and Pollina, 2000).
Urotensin II and Somatostatin
Urotensin II is secreted from the urophysis at rates responding to external
salinities with circulating levels being higher in seawater- than in
freshwater-adapted fish, suggesting a role in water retention (Bond et al.,
2002). In agreement with this putative role for urotensin II are
observations of increased short circuit current (depending on the dose) in
marine teleost intestinal epithelia conducted by apical Na+:Cl cotransport systems (Loretz et al., 1985; Baldisserotto and Mimura, 1997).
This stimulation of net salt absorption is very likely to facilitate water
absorption across the intestine epithelium. Interestingly, the partial
structural analog of urotensin II somatostatin alone has no effect on Na+
and Cl absorption (Loretz, 1990).
Cortisol
Cortisol is clearly involved in seawater adaptation by teleosts, as is evident
from increased plasma cortisol levels following transfer to seawater
(Mommsen et al., 1999) and pronounced effects on the gill chloride cells
and the mRNA expression of a number of ion-transporting proteins
(McCormick, 2001). The effects of cortisol enhance the ability of
350
Fish Osmoregulation
euryhaline fish to tolerate seawater exposure (Hwang and Wu, 1993) likely
via glucocorticoid receptors (Hirano, 1967; Hirano and Utida, 1968;
Gaitskell and Chester Jones, 1970; Epstein et al., 1971; Pickford et al.,
1979; Cornell et al., 1994; Marshall et al., 2005). Among its effects, cortisol
acts on the intestinal epithelium stimulating greater water absorption and
NKA activity (Cornell et al., 1994; Veillette et al., 1995). Thus, in contrast
to ANP, NPY and Urotensin II all of which act on apical transport proteins
in a rapid manner, cortisol promotes salt and water absorption by
increasing the NKA activity (presumably by increasing the gene
transcription and thereby amount of functional protein) in the basolateral
membrane.
Prolactin
Prolactin is critical for freshwater adaptation in the euryhaline killifish
(Pickford and Phillips, 1959) but, apparently, this is not a general
phenomenon for euryhaline fish (Takei and Loretz, 2005). The role of
prolactin (if any) in intestinal osmoregulatory function in seawater fish
remains unclear but prolactin appears to stimulate intestinal NKA activity
in fish held in both hypo-and hyper-osmotic media under certain
conditions (Kelly et al., 1999; Manzon, 2002).
Growth Hormone and Insulin-like Growth Factor
Growth hormone (GH) appears to play an important role in seawater
acclimation in teleost fish (Mancera and McCormick, 1998) and plasma
GH displays a transient increase following seawater transfer and protects
against increased plasma osmolality during seawater exposure (Sakamoto
et al., 1993). GH may act directly on osmoregulatory organs or by locally
secreted insulin like growth factor (IGF-1) but direct action of GH or IGFI on the osmoregulatory function of marine teleost intestinal epithelia
remains to be demonstrated.
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Fish Osmoregulation
+0)26-4
13
The Use of Immunochemistry in
the Study of Branchial Ion
Transport Mechanisms
INTRODUCTION
There are a number of recent reviews both in this volume and elsewhere
that address the role of branchial ion transport proteins in fish
ionoregulation (e.g., Cutler and Cramb 2001; Claiborne et al., 2002;
Marshall 2002; Hirose et al., 2003; Perry et al., 2003; Evans et al., 2005;
Galvez et al., 2007). Therefore, I intend to focus this short review on the
contribution of the immunological approach to our present state of
understanding. The advantages as well as shortcomings of the technique
and its contributions to our field will be discussed, in addition some
technical consideration will be reviewed. In Table 13.1, I have listed the
antibodies used in fish gill ionoregulatory research with their relevant
Authors address: Laboratrio de Ecofisiologia, Centro Interdisciplinar de Investigao
Marinha e Ambiental, Rua dos Bragas 289, 4050-123, Porto, Portugal.
E-mail: wilson_ jm@cimar.org
ICC, WB
aa 1377-1480 C-term fusion prot. (human)
ICC, WB
WB
WB
WB
WB
24-1(mo mc)
310 aa C-term NKCC1 (human)
ICC,
ICC
WB
ICC,
ICC,
ICC,
Full length CFTR recombinant prot. (human) ICC, WB
(mo mc)
aa 469-773 recombinant fragment (eel)

aa 469-773 recombinant fragment (eel)
125 aa fusion protein (tilapia)
Purified kidney a subunit (chicken)
Purified axolemma a3 (rat)
DAGKLQEVKYFGIGD aa 222-236 (eel b1)
ea NKA(rb pc)
ea NKA(rt pc)
TG3 (rb pc)
a6F(rb pc)
Ax2(mo mc)
b233(sh pc)
ICC, WB
TAM18 (mo mc)
CVTGVEEGRLIFDNLKKS
NAK121(rb pc)*
ICC
Format
Cystic fibrosis
transmembrane
regulator (CFTR)
Purified kidney a subunit (chicken)
a5(mo mc)
Antigen
T4
Antibody
Na :K :2Cl
cotransporter (NKCC)
Na +/K+-ATPase
Protein (subunit or
isoform)
F. heteroclitus
P. schlosseri
Periophthalmodon schlosseri
Anguilla anguilla
Anguilla japonica
Oncorhynchus masou
A. japonica
Tribolodon hakonensis
O. mossambicus
Oncorhynchus mykiss
Fish species
(Table 13.1 contd.)
13, (14)
10, (12)
10, (11)
4
5
6
7, (2)
7, (8)
9
1, (2)
Reference
Table 13.1 List of homologous and non-homologous antibodies used in the study of ion transport protein expression in the fish gill. The
studies cited are for first use only although many have gain widespread use (notably the a5, NAK121, T4 and 24-1 antibodies (see text for
additional citations)). The Table is organized into columns indicating the antibody target protein, subunit, or isoform specificity, the antibody
name with the host species (rb: rabbit, rt: rat, mo: mouse, or sh: sheep) and whether the antibody is monoclonal (mo) or polyclonal (pc),
the antigen the antibody was raised against (oligopeptide, purified or recombinant protein with origin), the format the antibody has been used
in: immunocytochemistry (ICC) and western blot (WB), the fish species studied, and the citation of the first fish study. If appropriate the
original source of the antibody is cited after in brackets.
360
Fish Osmoregulation
Na /H exchanger
(NHE)
CNRNVELNGPEAAARQHAQA (eel B1)

CGANANRLFLD (bovine, Hemken
et al. 1992)**
CGANANRLFLD (bovine)
1035(rb pc)
E(rb pc)
E11 (mo mc)
B1
E(31 kDa)
597(rb pc)
2M5(rb pc)
1380(rb pc)
2
3
4E9
87aa C-term fusion prot. (rabbit)

aa 260-280 fusion prot. (rat)
85aa C-term fusion prot. (rabbit)
aa 514-818 C-term fusion prot. (pig)
RKDHADVSNQLYACYA (eel B1/B2)
B2/BvA1(rb pc)
(mo mc)
160aa recombinant protein (partial)(dace)

[LPDGTKRSG]-MAP4 (eel B1/B2)
CSHITGGDIYGIVNEN (bovine)
CAEMPADSGYPAYLGAR (killifish)
279aa recombinant prot. (aa 79-357)
(mosquito)
dB(rb pc)
1034(rb pc)
130 aa fusion prot. (aa 124-254) (eel)
A(70 kDa) A(rb pc)

kf A(rb pc)
B (56kDa) B(rb pc)
V-ATPase
Potassium Channel (Kir) eKir(rb pc)
(Table 13.1 contd.)
ICC, WB
WB
ICC, WB
WB
ICC, WB
WB
ICC, WB
ICC
WB
WB
ICC, WB
ICC, WB
ICC, WB
ICC, WB
F. heteroclitus,
Myoxocephalus
octodecimspinosus
P. schlosseri
F. heteroclitus
O. mykiss, Pseudolabrus
tetrious
Geotria australis
A. anguilla
Polypterus senegalus
Acipenser transmontanus
D. rerio
O. mykiss
T. hakonensis
Danio rerio
O. mykiss
Fundulus heteroclitus
Dasyatis sabina
A. japonica
15
(Table 13.1 contd.)
10, (29)
30, (31)
32, (33)
27, (28)
25, (26)
21, (22)
24
23
5
21, (22)
16, (17)
18
19, (20)
361
(rb pc)
(rb pc)
rtECaC (rb pc)
Epithelial Calcium
Channel (ECaC)
(mo mc)
5F10
SQFRFRLQNRKGWKEMLD (aa 18-36)

(trout)
Purified erythrocyte PMCA (human)
42 aa COOH term fragment (dace)
HKa1 (C2) (rb pc) GVRCCPGSWWDQELYY (pig)
Ca2+-ATPase (PMCA)
ICC, WB
WB
ICC, WB
WB
ICC
ICC, WB
WB
O. mykiss
Oncorhynchus neta
ICC, WB
ICC, WB
Protopterus annectens
D. sabina
T. hakonensis
D. sabina
O. mossambicus
O. mykiss,
O. mossambicus
O. mossambicus
O. mossambicus
F. heteroclitus
Raja erinacea
T. hakonensis
D. sabina
ICC
ICC, WB
ICC
PTKEIEIQVDWNSE aa630-643 (human/rat) ICC, WB
H+/K+-ATPase
(rb pc)
dNBC1
(rb pc)
Purified erythrocyte AE1 (trout)
Amiloride-sensitive Na + channel (bovine)
GEKYCNNRDF aa 411-420 (human)
Full length fusion protein (bovine)
DASVDEEASEEKPGKNHTRL (dace)
212aa C-term recombinant fragment (ray)
aa 528-648 fusion prot. (rat)
Na +: HCO 3
cotransporter (NBC)
h630-643
Pendrin
(rb pc)
Hillary(rb pc)
55-1
(rb pc)
AE1t
a 1190-2
dNHE3 (rb pc)

R1B2 (rt pc)
666
Anion Exchanger 1
Epithelial Sodium
Channel (ENaC)
(Table 13.1 contd.)
(Table 13.1 contd.)
49
48, (47)
46, (47)
44, (45)
42, (43)
37, (41)
37, (40)
37, (39)
37, (38)
5
36
34, (35)
362
Fish Osmoregulation
208aa recombinant fragment N-term (eel)
eUT1(rb pc)
Urea transporter (UT)
ICC, WB
ICC, WB
ICC
ICC, WB
ICC, WB
ICC, WB
ICC
A. japonica
A. anguilla
T. hakonensis
O. mossambicus
O. mykiss
O. mykiss
T. hakonensis
54
52
5
53
51
50
5
** citation in parentheses refers to origin of peptide sequence (non-fish)

1) Witters et al. 1996; 2) Takeyasu et al. 1988; 3) Ura et al. 1996; + 4) Mistry et al. 2001; 5) Hirata et al. 2003; 6) Hwang et al. 1998; 7) Lee et al. 1998; 8) Sweadner and Gilkeson
1985; 9) Cutler et al. 2000; 10) Wilson et al. 2000b; 11) Lytle et al. 1995; 12) Neomarkers; 13) Marshall et al. 2002; 14) R&D Systems; 15) Suzuki et al. 1999; 16) Lin et al. 1994;
17) Sudhof et al. 1989; 18) Katoh et al. 2003; 19) Piermarini and Evans 2001; 20) Filippova et al. 1998; 21) Boesch et al. 2003b; 22) Boesch et al. 2003a; 23) Wilson (this article);
24) Sullivan et al. 1996; 25) Choe et al. 2004; 26) Hemken et al. 1992; 27) Claiborne et al. 1999; 28) Rutherford et al. 1997; 29) Tse et al. 1994; 30) Edwards et al. 2005; 31) Bookstein
et al. 1997; 32) Edwards et al. 1999; 33) Hoogerwerf et al. 1996; 34) Chloe et al. 2002; 35) Soleimani et al. 1994; 36) Choe et al. 2005; 37) Wilson et al. 2000a; 38) Ismailov et al.
1996; 39) D. Benos, unpublished; 40) Sorscher et al. 1988; 41) Cameron et al. 1996; 42) Piermarini et al. 2002; 43) Royaux et al. 2000; 44) Choe et al. 2004; 45) Smolka and Swinger
1992; 46) Sturla et al. 2001; 47) Borke et al. 1989; 48) Uchida et al. 2002; 49) Shahsavarani et al. (in press); 50) Rahim et al. 1988; 51) Georgalis et al. 2006; 52) Lignot et al. 2002;
53) Watanabe et al. 2005; 54) Mistry et al. 2001.
* the peptide originally designed by Ura has also been used by Uchida et al. (2000) to make a anti-peptide polyclonal antibody they call NAK121 that is cross reactive with the
denatured protein in Western blots. Ura et al. (1996) reported cross reactivity only with the native protein in Western blots. Katoh et al. (2000) have also affinity purified and directly
labeled this antibody with FITC. I have also generated rabbit polyclonal antibodies using the same peptide sequences and refer to this antibody as aRbNKA.
DERIKLSNVATKDAA (eel)
DKTNKDMEESLKLNDVTGKN (dace)
HTEGEARDKKQGT (tilapia)
Purified gill CA (trout)

260aa recombinant protein
(full length) (dace)
WNTKYPSFGDAASKSDGLA (trout)
eAQP-3
dAQP-3(rb pc)
tilAQP-3(rb pc)
(rb pc)
tCAc(rb pc)
CAB(rb pc)
dCAII(rb pc)
Aquaporin 3
Carbonic anhydrase II
(Table 13.1 contd.)
363
364
Fish Osmoregulation
information (target protein, antibody name, antigen, species, and

references). From this Table, the wide use of this technique in our field is
quite evident. This short review will also focus on immunocytochemistry
(ICC) and will only make reference to immunoblotting, although in itself,
it has proven to be a very useful technique.
The anatomical complexity of the fish gill (Wilson and Laurent, 2002)
has hampered our progress in the elucidation of the molecular mechanisms
involved in ion regulation (see the reviews listed above). This has been
largely due to the unsuitability of the gills for use in ussing chamber type
experiments that allow a detailed study of ion movements but are suited
to flat two-dimensional epithelia (see Wood et al., 2002). The study of
freshwater ion uptake mechanisms, in particular, has lagged behind due to
the lack of suitable surrogate models that can be studied with such a
system. In the case of the seawater fish, the opercular epithelium
preparation has served this role (see Marshall, 2002).
The immunological approach to the study of ion transport protein
expression has allowed us to identify the location of many ion transport
proteins and infer their function from this localization. This is a powerful
means of conveying such information as the adage goes a picture speaks
a thousand words. For a verbally handicapped person such as myself, this
technique has been a particular godsend. It is essential, however, that
proper controls are run for the correct interpretation of the results (see
below).
Although antibodies are well suited as probes for determining the
location of proteins of interest, they do have certain obvious limitations.
The most notable is that they cannot tell us whether the protein is indeed
functional (active). The nature of the specificity is immunologic and not
biologic (Petrusz et al., 1976). The activities of many proteins are regulated
by a number of different factors that would not be reflected in the
antigenicity of that protein. There are, however, some antibodies that do
recognize epitopes in key regulatory sites and only bind when the protein
is activated (Flemmer et al., 1999). One simple way to overcome this
shortcoming is to use ICC in conjunction with physiological techniques
from which the function can be ascertained.
Another problem relates to the use of antibodies in comparative
studies. The major difficulty comes in explaining the significance of a
negative result since there are a number of explanations for differences in
365
strength of immunoreactivity between different species. These could

include difference in expression levels and/or distribution, to varying levels
of antibody specificity due to differences in the antigenic sites which affect
antibody binding and, consequently, signal strength. In general, positive
control tissue should be used when assessing antibody labeling with a new
species, organ or antibody in order to rule out faults with the protocol. The
a5 monoclonal antibody, which is generally considered pan-specific
(recognizes a very conserved epitope of the Na+/K+-ATPase a subunit),
has been reported to work in a wide variety of organisms, including
numerous fishes. However, in the small spotted cat shark, Scyliorhinus
canicula, I was unable to get cross-reactivity with this antibody in gill,
kidney or rectal gland with either ICC or Western blotting. The presence
of Na+/K+-ATPase was confirmed by measuring in vitro ouabain-sensitive
activity and by using the aRbNKA antibody (which also recognized a
highly conserved region of Na+/K+-ATPase a subunit) with both ICC and
Western blots. Care must, therefore, be taken with the use of this
technique in comparative studies and alternative techniques used for
confirmation. Generally, it is advisable to use more than one antibody to
the protein of interest, if possible (Nigg et al., 1982).
ADDING TO THE SEAWATER MODEL OF ION
EXCRETION
The components of secondary active chloride secretion identified using
electrophysiological and pharmacological techniques have all been
identified and localized by immunochemistry (Marshall, 2002). The
basolateral Na+/K+-ATPase creates a Na+ gradient to drive Cl uptake via
the Na+:K+:2Cl cotransporter (basolateral isoform NKCC1) into the
cell, which accumulates and exits the cell apically via a cystic fibrosis
transmembrane regulator (CFTR) Cl channel down its electrochemical
gradient. These transporters have all been localized to the mitochondriarich chloride cell in the gill (e.g., Wilson et al., 2000b; McCormick et al.,
2003), opercular epithelium (Marshall et al., 2002) and yolk sac epithelium
(Hiroi et al., 2005). Figure 13.1 illustrates the colocalization of NKCC and
Na+/K+-ATPase to the basolateral tubular system and the localization of
CFTR to the apical crypts of these cells in gill sections of seawater pipefish
(Syngnathus). In addition to confirming the presence of ion transport
proteins in the seawater chloride cells, more recently, it has been
demonstrated that the cellular distribution of CFTR and NKCC change
366
Fish Osmoregulation
Fig. 13.1 Indirect immunofluorescent labeling of pipefish (Syngnathoidei) gill filament

transverse sections for the ion transport proteins involved in secondary active Cl secretion.
Sections are double labeled for (a) CFTR and (a) Na+/K+-ATPase or (b) NKCC and (b)
Na+/K+-ATPase, with corresponding DIC images for tissue orientation (a, and b
respectively). The insets provide higher magnification (4 ) of the CFTR apical crypt
staining of Na+/K+-ATPase immunoreactive cells as indicated by the arrows. CFTR and
NKCC were immunolocalized with monoclonal antibodies antibody 24-1 (R&D Systems),
and T4 (DSHB), respectively and Na+/K+-ATPase with the rabbit anti-peptide polyclonal
antibody aRbNKA. Scale bar = 100 mm (J.M. Wilson and P. Laurent, unpublished
observations).
367
with salinity (Marshall et al., 2002; Hiroi et al., 2005). Hiroi and coworkers working with tilapia larvae yolk sac epithelium (2005) have
employed direct triple labeling of these three transporters to identify four
subtypes of mitochondria-rich cell (MRC) [type I: basolateral Na+/K+ATPase only; type II: basolateral Na+/K+-ATPase and apical NKCC
(apical isoform NKCC2); type III: apical CFTR and basolateral Na+/K+ATPase and NKCC1 and type IV: basolateral Na+/K+-ATPase and
NKCC1]. The relative abundance of these MRC sub-types changes with
both freshwater and seawater adaptation, indicating a role of the type III
cell in seawater and the type II in freshwater ion regulation. The apical
NKCC2 will be discussed further in the following sections on freshwater
ion uptake mechanisms.
The accessory cell, which is found to be closely associated with the
chloride cell, is an integral part of the seawater fish ion regulatory model
because the tight junctions it forms with the chloride cell are leaky (see
Wilson and Laurent, 2002). These leaky junctions provide the paracellular
pathway for Na+ to pass down its electrochemical gradient out of the fish.
In the coho salmon, the NHE2 antibody 597 (Tse et al., 1994) specifically
labelled cells that, based on their morphology and appearance, appear to
be the accessory mitochondria-rich cell (Wilson et al., 2002b). These cells
are slender and juxtaposed to Na+/K+-ATPase rich chloride cells and
extend apically into the crypt. A similar staining pattern is observed in
tilapia (O. mossambicus) acclimated to seawater (Fig. 13.2). In freshwateracclimated tilapia, the frequency of these cells is reduced and there is an
absence of CC-AC apical crypts. Although it is unclear why NHE-2
immunoreact with AC in this way (see Wilson et al., 2002b), the antibody
597 may prove to be a useful tool for studying ACs in much the same way
that some antibodies are used as marker to identify different cell types.
FRESHWATER ION UPTAKE MECHANISMS
Sodium Uptake Mechanism
One on-going debate that immunochemistry seems well suited to address
is the relative importance of direct versus indirect coupling of Na+ and H+
exchange in acid-base regulation and sodium uptake. The first proposed
mechanism involves direct coupling via a sodium-proton exchange (NHE)
protein which utilizes either a sodium or proton gradient to drive the
exchange. The sodium driven exchange would be predicted to function in
368
Fish Osmoregulation
Fig. 13.2 Photomicrograph of NHE-2 (Ab597) colabeling with Na+/K+-ATPase (a5) in

freshwater (a and a, respectively) and seawater (b and b, respectively) adapted tilapia (O.
mossambicus) gill. The corresponding phase contrast images (a and b). Arrows indicate
location of NHE-2 immunoreactive cells in freshwater tilapia. Scale bar = 10 mm (J.M.
Wilson and T.J. Lam, unpublished observations).
acid excretion, while the H+ driven exchange would facilitate Na+ uptake.
The second proposed mechanism involves indirect coupling via an apical
proton pump (vacuolar type proton ATPase) and a sodium channel,
whereby the proton pump creates the favourable electrochemical gradient
for sodium uptake against its concentration gradient. It should be noted
that these two mechanisms need not be mutually exclusive, as is evident
in the mammalian kidney (NHE in the proximal tubule and V-ATPaseENaC in the collecting duct).
The emerging picture in fishes appears to be that the dominant
mechanism is, in part, determined by the origins of the species with marine
euryhaline fishes favouring NHE (sculpin, killifish and elasmobranchs)
and euryhaline freshwater fishes favouring the V-ATPase (predominantly
the salmonids). The acid-tolerant dace would be an exception since the
NHE is responsive to acid exposure, while the V-ATPase is not. This may
be a result of its adaptation to a very unusual environment (Hirata et al.,
2003). In the case of the NHE, there are also differences in expression of
isoforms with NHE2 being upregulated in the freshwater-adapted killifish
(F. heteroclitus, Edwards et al., 2005), while the NHE3 is more important in
the freshwater-adapted Atlantic stingray (Choe et al., 2005), acid-tolerant
dace (Hirata et al., 2003) and seawater-adapted killifish (Edwards et al.,
2005). The killifish NHEs are responsive to environmental hypercapnia
369
(1% CO2) (Edwards et al., 2005). However, in the Atlantic stingray, they
are not (Choe et al., 2005). It would, thus, appear that the physiological
roles of the NHEs are not uniform in fishes.
In dace, V-ATPase expression reported using Western analysis
indicates that it is unresponsive to environmental pH, while NHE3 is
strongly upregulated under acidic conditions (Northern analysis). The
apical immunolocalization of proton-ATPase has been demonstrated in
the gills of a number of different species of fish (salmonids (trout and
salmon), tilapia, zebrafish) (e.g., Lin et al., 1994; Sullivan et al., 1995,
Wilson et al., 2002b). In the grey bichir, Polypterus senegalus senegalus, VATPase is found apically in a cell type distinct from those rich in Na+/K+ATPase (Fig. 13.3). In the freshwater loach (Misgurnus anguillicaudatus), a
similar staining pattern has been observed (J.M. Wilson, unpublished
observations).
There is only one report of the presence of a sodium channel (ENaC,
epithelial sodium channel) in trout and tilapia gill using a battery of nonhomologous antibodies directed against mammalian ENaC (Wilson et al.,
2000a). This cross-reactivity is supported by the finding of phenamil
(ENaC specific inhibitor) sensitive Na+ uptake but not by molecular
genetic analyses (Perry et al., 2003). It is, thus, uncertain based on
immunochemical results whether the V-ATPases localized in fish gill are
driving Na+ uptake via an epithelial type Na+ channel.
Recently, the NKCC has entered the debate after being
immunolocalized to the apical membrane of freshwater Na+/K+-ATPase
immunoreactive cells in tilapia (O. mossambicus) using the T4 antibody
(Wu et al., 2003; Hiroi et al., 2005). To support a possible role for the
NKCC2 (apical isoform) in Na and Cl uptake is the finding of furosamide
sensitivity in the goldfish (Preest et al., 2005). However, I have been
unsuccessful in immunolocalizing an apical NKCC in goldfish (J.M.
Wilson, unpublished observations). Although in the climbing perch (A.
testudineus) gill, I have been able to find apical NKCC immunoreactivity
associated with Na+/K+-ATPase immunopositive cells (Fig. 13.4). In the
case of the goldfish, it has long been known that Na and Cl uptake can
operate independently (Maetz and Garcia-Romeu, 1964), which would
indicate that the NKCC is not the only mechanism present. Hiroi et al.
(2005) have made preliminary reports on the presence of both NKCC1
and NKCC2 mRNA in tilapia gill. In killifish, the NKCC2 mRNA
expression has been reported as low but detectable in gill although not
370
Fish Osmoregulation
Fig. 13.3 Immunolocalization of (a) Na+/K+-ATPase (a5 antibody) and (a) V-ATPase (B2
antibody) in the grey bichir, Polypterus senegalus senegalus, gill filament sagittal section
by double immunofluorescence microscopy. (a) The corresponding differential
interference contrast (DIC) image. Arrows indicate the apical V-ATPase immunolabeling
and asterisks are placed over the nuclei of Na+/K+-ATPase immunoreactive cells. Scale bar
= 25 mm (J.M. Wilson and Y.K. Ip, unpublished observations).
modulated by salinity (Scott et al., 2005). The NKCC2s relative

importance in Na, K and Cl uptake remains to be determined as well as the
gradients responsible for driving cotransport.
371
Fig. 13.4 Immunolocalization of NKCC (T4 antibody) to the apical region of Na+/K+ATPase immunoreactive cells (aRbNKA antibody) in the gills of the climbing perch (Anabas
testudinus). Arrows indicate the apical NKCC labeling. No basolateral labeling was
observed. Scale bar = 50 mm (J.M. Wilson and Y.K. Ip, unpublished observations).
CHLORIDE UPTAKE
Chloride uptake in freshwater fishes is largely believed to be facilitated by
a SITS sensitive electroneutral Cl/HCO 3 anion exchanger (AE; e.g.,
reviewed by Evans et al., 2005). The molecular identification of the AE in
teleost fishes has been elusive. Apical AE1 (band 3) immunoreactivity has
been reported in the tilapia and coho salmon associated with a
subpopulation of Na+/K+-ATPase immunopositive cells (Wilson et al.,
2000a, 2002b) using an antibody developed against trout band 3 protein
372
Fish Osmoregulation
(Cameron et al., 1996). In coho salmon, this apical immunoreactivity is

not present in seawater-adapted fish. However, the only supporting data
for this type of exchanger is an in situ hybridization using an
oligonucleotide probe in trout gill (Sullivan et al., 1996). In the euryhaline
Atlantic stingray (Dasyatis sabina), apical immunoreactivity to pendrinlike protein (a Cl/HCO3 anion exchanger) in cells with basolateral VATPase expression (Piermarini et al., 2002). The basolateral V-ATPase
helps drive the apical exchange by removing H+ from the cell in order to
maintain the intracellular HCO 3 which directly drives the exchange.
Another proposed AE in the gill is the putative anion transporter 1
(PAT1) that has been identified in trout gill. However, additional
characterization is still awaited (G.G. Goss U. Alberta, unpublished
observations). In the killifish, V-ATPase (A subunit) has been
immunolocalized to gill Na+/K+-ATPase immunoreactive cells
(mitochondria-rich cells) in a basolateral location and was suggested to
play a similar role as in the Atlantic stingray (Katoh et al., 2003). However,
the killifish Cl/HCO 3 exchange has not been found to be of physiological
significance and, thus, the role of basolateral V-ATPase in the killifish is
somewhat unclear (Kirschner, 2004). The NKCC2 also represents another
pathway for Cl uptake to be further explored (Hiroi et al., 2005; Preest
et al., 2005).
OTHER TRANSPORTERS IN THE GILL
In addition to the ion transport proteins involved in Na+ and Cl
regulation, a number of other transporters have been recognized in the
gills of fishes (see Table 13.1). Of note are the urea transporter (UT; Mistry
et al., 2001) and aquaporin 3 (water channel proteins; Lignot et al., 2002;
Hirata et al., 2003; Watanabe et al., 2005) which are both found localized
to the mitochondria-rich cells tubular system (basolateral membrane).
The plasma membrane calcium ATPase (PMCA) is also found associated
with the tubular system (e.g., Sturla et al., 2001; Uchida et al., 2002) and
an epithelial calcium channel (ECaC) with the apical membrane
(Shahsavarani et al., in press).
IDENTIFICATION OF NA+/K+-ATPASE
IMMUNOREACTIVE CELLS. CHLORIDE CELLS?
Immunocytochemistry has become almost a standard technique to study
the branchial mitochondria-rich chloride cell. This is largely due to the
373
high abundance of Na+/K+-ATPase associated with either the tubular

system or basal labyrinth or these cells and, most importantly, a source of
very specific and inexpensive anti-Na+/K+-ATPase antibody. The cellular
immunolabeling pattern is characteristic of the MRC type, thus making it
readily identifiable for enumeration and characterization (size, shape
factor, location). The bulk of these studies have made use of the a5 mouse
monoclonal antibody first developed by Douglas Fambrough and available
though the Developmental Studies Hybridoma Bank at a very reasonable
price (currently 25$ per ml culture supernatant). The a5 antibody
recognizes an epitope in a conserved region of the a subunit and is not
isoform specific (5 a subunit isoforms have been characterized so far).
Another antibody that has also been widely usedalthough is not so
readily availableis the anti-peptide antibody initially developed by Ura
et al. (1996) and used more extensively by the group of Kareko (e.g. Uchida
et al., 2000; Katon et al., 2000) and given the name NAK121. I have raised
antibodies against the same peptide (aRbNKA).
Na+/K+-ATPase immunoreactive cells have been identified in a wide
range of fish, including hagfish (Myxine glutinosa Choe et al., 1999),
lamprey (Geotria australis Choe et al., 2005), ray (Dasyatis sabina
Piermarini and Evans, 2000), shark (Squalus acanthias Wilson et al., 2002)
as well as a plethora of teleost fishes (e.g., Anguilla japonica, Oncorhynchus
masou Ura et al., 1996; O. mykiss Witters et al., 1996; Fundulus heteroclitus
Katoh et al., 2000; Oreochromis mossambicus Dang et al., 2000;
Periophthalmodon schlosseri Wilson et al., 2000b; Stenogobius hawaiiensis
McCormick et al., 2003). Lee et al. (1998) have also used other antibodies
against specific a-subunit isoforms that have been found to localize to
MRCs in the tilapia (O. mossambicus) while Hwang et al. (1998), Cutler
et al. (2000) and Mistry et al. (2001) have developed homologous
antibodies in O. mossambicus (tilapia), A. anguilla (European eel) and A.
japonica (Japanese eel), respectively.
The immunological approach has largely surpassed other techniques
that target Na+/K+-ATPase (3[H] ouabain autoradiography and
anthroylouabain fluorescence, histochemistry), or the membrane systems
(Champy-Malliet stain) of MRCs (see Wilson and Laurent, 2002). The
main advantage to this system of MRC identification over mitochondrial
vital stains such as DASPMI (dimethylaminostyryl-methylpyridiniumiodide, emission wavelength 609 nm) and DASPEI
(dimethylaminostyrylethylpyridiniumiodide, emission wavelength 530nm)
374
Fish Osmoregulation
(e.g., Li et al., 1995; Witters et al., 1996; Rombough, 1999) and

Mitotracker (Molecular Probes, Galvez et al., 2002; Marshall et al., 2002)
is that fixed and processed tissue sections can be used. Both types of
mitochondrial stain require live mitochondria, although in the case of
Mitotracker, the cells can be post-fixed. There have only been a few studies
using immunohistochemical techniques to detect mitochondria using
antibodies directed against mitochondria specific proteins (HSP 60,
Mickle et al., 2000; Ab-AMA, Sturla et al., 2001).
In addition to counting the immunopositive cells, the signal strength
(intensity), area of immunolabeling, and cell shape factor, can be measured
and cellular expression calculated (area intensity) using commercially
available image analysis software or freeware (NIH image). Commonly,
counting of cells is complicated by the fact that MRCs are not evenly
distributed across the filament being more abundant towards the afferent
side. Counting is, therefore, usually done at the afferent end. Counts are
also commonly separated into lamellar and filament epithelial populations
of cells (e.g., Pelis et al., 2001).
Abundance of Na+/K+-ATPase has most commonly been measured
biochemically using ouabain as the specific inhibitor of Na+/K+-ATPase
activity in vitro under optimal conditions (Vmax; e.g., McCormick et al.,
1993). However, more recently, Western blotting has also been used to
quantify Na+/K+-ATPase subunit abundance. Generally, there is a good
correlation with the two approaches (Piermarini and Evans, 2000; Wilson
et al., 2004).
TECHNICAL CONSIDERATION
In the second half of this review, I will cover some technical aspects of
immunochemistry and also analyse the application of methods in fish gill
research.
THE ANTIBODY
The use of antibodies as a tool to localize molecules of interest in
microscopy was first demonstrated in the 1940s (Coons et al., 1940), with
immunohistochemcial techniques coming into broader use in the 1970s
(Taylor et al., 1974). It is widely appreciated that antibodies can make
excellent probes for detecting proteins of interest. The acquired immune
response is hijacked to produce specific antibodies to proteins (or other
375
antigens) (whole or in part; biochemically purified or synthesized) injected

into a host animal. With subsequent boost injects, the levels of the
polyclonal antibodies in the blood increases and can easily be collected by
bleeding the host animal and separating the serum. The antibodies can
then be further purified. Alternatively, hybridoma techniques allow the
immortalization of B cells in order to produce monoclonal antibodies
either in vivo or in vitro. It is beyond the scope of this review to elaborate
further as this topic is dealt with in textbooks. One important point to keep
in mind is that a monoclonal antibody recognizes a single epitope, while a
polyclonal antibody recognizes multiple epitopes.
To my knowledge, the first such study to apply an immunological
approach in the study of the fish gill was by Rahim and co-workers (1988)
in the study of trout carbonic anhydrase. Having found that commercially
available antibodies raised against mammalian erythrocyte CA were not
cross reactive in the trout, they proceeded to biochemically purify gill and
erythrocyte CA and generate homologous antibodies (P. Laurent, pers.
comm.). Their findings have remained significant as CA which reversibly
catalyzed CO2 hydration is important in providing the intracellular
counter ions, H+ and HCO 3, for Na+ and Cl exchange, respectively.
Research of fish gill ion transporters has, however, relied on the use of nonhomologous mammalian antibodies largely due to their availability and
species cross-reactivity (see Table 13.1). This indicates the conserved
nature of some of these proteins. However, more recently, there has been
a greater tendency to developing homologous antibodies for fish studies
and approximately half of the antibodies listed in Table 13.1 are
homologous for the species being studied.
In many recent reviews, it is common to see that future work should
involve the development of homologous antibodies. Although, this is
ultimately desirable it is not, in my opinion, always necessary. The most
notable examples are the a5 and Ura antibodies that can be considered
pan-specific for the a subunit of Na+/K+-ATPase as the epitopes for these
antibodies are conserved in (most) vertebrates and invertebrates (the
notable exception being S. canicula as discussed earlier). They have
consequently enjoyed widespread use with over 50 publications in the fish
gill literature. They have proved invaluable in characterizing the dynamics
of mitochondria-rich (Na+/K+-ATPase immunoreactive) cells in response
to environmental change as well as for determining the presence of
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Fish Osmoregulation
Na+/K+-ATPase-IR cells in less studied species. The T4 monoclonal

antibody directed against NKCC (isoforms 1 & 2) is also widely used
although some immunostaining results require further evaluation.
However, the study of Na+/K+-ATPase will need to progress beyond the
use of isoform non-specific antibodies as recent studies have indicated the
importance of isoform switching in adaptation (a1a and a1b; Richards
et al., 2004).
In addition, there are other commercially available monoclonal
antibodies that recognize conserved epitopes of plasma membrane Ca2+
ATPase (PMCA; 5F10 (Sturla et al., 2001; Uchida et al., 2002) and CFTR
(24-1) (Marshall et al., 2002). In the case of antibodies generated against
short peptides, it is possible in many cases to compare the peptide
sequence to those available on-line (genebank) using a Blast-p search.
Examples of this form of validation can be found in the work of Edwards
et al. (2005) using mammalian NHE2 antibodies and Wilson et al. (2000a)
for V-ATPase A subunit. From this information, it is possible to determine
the conserved nature of the peptide sequence and also the likelihood that
the cross reactivity is indeed specific. In the cases where sequence
information for the antigen of non-homologous antibodies is not known,
some caution should be taken in interpreting the results in the absence of
strong supporting evidence.
With advances in PCR-based cloning techniques and with sequence
information readily available through the Internet, it is now reasonable for
most laboratories using this information to deduce the amino acid
sequences in order to have antigens for antibody production produced
either through solid state peptide synthesis or recombinant protein
synthesis. An impressive example is the recent publication by Hirata et al.
(2003) on the acid-tolerant dace in which four ion transport proteins are
cloned and homologous antibodies generated (V-ATPase B, NHE 3, AQP
3, CAII).
FIXATION
Fixation serves to preserve the structural integrity of the tissue and to
insure that cellular components remain in place. Good fixation generally
comes at a cost to tissue antigenicity and a compromise must be made.
In the existing fish gill literature, a number of different fixatives have
been used successfully for ICC. The most common fixatives are
377
paraformaldehyde (PFA) based (3-4%) in a phosphate-buffered saline

(PBS; pH 7.3-74) (e.g., Uchida et al., 1996; Edwards et al., 1999; Seidelin
and Madsen, 1999). Glutaraldehyde is added to improve fixation although
generally at low concentrations 0.05% to 0.1% (e.g., Rahim et al., 1988;
Sullivan et al., 1995; Piermarini and Evans, 2000) with the exception of
Lee et al. (1998; 1%). Generally, tissue fixation is adequate with PFA at the
light microscopy level and glutaraldehyde, which is a better crosslinking
agent, may be associated with epitope masking so is best avoided.
However, for electron microscopy the inclusion of glutaraldehyde in the
fixative may become necessary for preservation of subcellular detail. Picric
acid, which is good at preserving membrane, is commonly used either as
Bouins solution (Wilson et al., 2002b) or as a 0.05% additive to PFA/PBS
(Sullivan et al., 1995; Piermarini and Evans, 2000).
Paraformaldehyde works by reacting with primary amines of lysine to
establish a cross linking. Fixation may prevent antibody binding to its
epitope by denaturing the epitope through its cross linking action or by
blocking access to the epitope by crosslinking neighbouring proteins.
However, it should be noted that not all epitopes are adversely affected by
fixation.
Tissue can also been fixed by protein precipitation with alcohols at low
temperature (ice cold EtOH: Witters et al., 1996; 20% DMSO in MeOH
at 20C: Pelis et al., 2001; Marshall et al., 2002; Wilson et al., 2004). The
DMSO is added to prevent ice crystal formation that would damage the
tissue. This technique avoids chemical cross linking but may still denature
the epitope and block access of the antibody. I have also noticed that with
20% DMSO in MeOH in some tissue preparations fixation artifact results
from the cytoplasmic content of cells being washed out. This artifact,
however, is easily identifiable and tissue preservation is generally good
since in the case of the gill, the diffusion distances are short. In direct
comparison with PFA fixed tissue, tissue antigenicity is markedly stronger.
EMBEDDING
Embedding of fixed tissue in paraffin or cryo-embedding (OCT
compound) for sectioning or whole mounting are commonly used. Both
cryosectioning and whole mounting tissue avoids additional harsh
treatment of the tissue that may further compromise tissue antigenicity.
Generally, immunoreactivity is better in tissue that has seen the least
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Fish Osmoregulation
processing with cryosections giving superior signal strength in direct

comparisons with paraffin embedded tissue. However, paraffin-embedded
tissue has the benefit of being easier to handle and archive as no special
conditions are required (room temperature). There are a number of
antigen retrieval or unmasking techniques that can sometimes be used to
recover tissue antigenicity in order to make results with paraffin embedded
tissues comparable to cryosections (see below).
DETECTION
A direct or indirect detection method is necessary in order to visualize the
antibody bound to the section through a marker molecule. The markers
commonly used are fluorochromes (e.g., FITC, TRITC, Cy3, Alexa Fluor
dyes), an enzyme that catalyzes a chromogenic reaction (horseradish
peroxidase (HRP) or alkaline phosphatase (AP)), or a gold particle. Direct
labeling involves the conjugation of the marker directly to the antibody
which results in lower background staining but requires that the antibody
be purified. The tissue staining pattern must also be well characterized (by
indirect detection) beforehand since it is difficult running negative
controls with direct labeling. Consequently, there have been very few
studies using direct labeling of antibodies with the exception of the group
of Kaneko (e.g., Katoh et al., 2000; Katoh and Kaneko, 2003; Hiroi et al.,
2005).
Practically all studies in the fish gill literature make use of indirect
detection, whereby the marker molecule is conjugated to a secondary
reagent that will recognize the primary antibody. This reagent is most
commonly a labeled species-specific anti-immunoglobulin antibody (for
example, when using a rabbit polyclonal primary antibody, a goat antirabbit IgG HRP conjugated secondary antibody is used). Protein A and G
(bacterial cell wall proteins) can also be used as secondary reagents
although their use is less common (Laurent, unpubl.). In addition to being
more sensitive, indirect labeling also allows for additional signal
amplification (ABC, BSA, PAP techniques see below).
The choice of marker molecule is determined in part by equipment
availability and the degree of resolution required. The use of fluorochrome
conjugates requires a fluorescence microscope and appropriate filter sets
but offers high resolution and the ability to use multiple labels (2-5) on the
same section. There are many examples of the use of fluorescence for
379
single antibody labeling (e.g., Lin et al., 1994; Sullivan et al., 1995; Lignot
et al., 2002). Double labeling of sections using indirect detection has been
done using a combination of mouse monoclonal and rabbit polyclonal
primary antibodies [a5 with V-ATPase A, tAE1, 597, or 1380 (Wilson
et al., 2000a, b, 2002b), NAK121, and T4 (Pelis et al., 2001); NAK121,
and kfA (Katoh et al., 2003); NAK121 and CFTR 24-1 (Katoh and
Kaneko, 2003); NAK121 and CFTR 24-1 or T4 (McCormick et al., 2003),
and eaNKA and NBC1 with the addition of a nuclear stain (Hoechst
33342; triple staining Hirata et al., 2003). The study of Hiroi et al. (2005)
is the first to use more than two antibodies at a time with direct labeling
[aNKA (NAK121) Alexa Fluor 546; NKCC (T4) Alexa Fluor 647 and
CFTR (24-1) Alexa Fluor 488]. Although this study is not on gill
epithelium, it does nicely illustrate the usefulness of direct labeling in
multiple labeling experiments.
The most commonly used enzyme conjugate is horseradish peroxidase
(HRP). It gives a stronger signal in a shorter period of time than alkaline
phosphatase, another enzyme conjugate which is not widely used in fish
ICC. Another advantage of using HRP is that the section can also be
permanently mounted. The most common chromagenic substrate used is
DAB (3,3-diaminobenzidine), which gives a brown-black reaction
product. In some double labeling experiments with HRP, Vector VIP
(purple; Vector Lab USA) or Vector SG (blue) have been used (Piermarini
et al., 2002; Choe et al., 2005). Although HRP can be used as a conjugate
to the secondary antibody (Wilson et al., 2000b; Hirata et al., 2003), it is
more commonly employed after a biotin amplification step to improve
sensitivity (biotin labeled species-specific secondary antiimmunoglobulin). Avidin and streptavidin both bind to biotin with high
affinity. Some examples of the use of the ABC (avidin biotin conjugate)
method include Uchida et al. (1996), Witters et al. (1996), Hiroi et al.
(1998), Suzuki et al. (1999), Katoh et al. (2000), while the BSA (biotin
streptavidin) method has been used by Cutler et al. (2000) and Piermarini
and Evans (2000). Another signal amplification technique employs the
use of an anti-peroxidase antibody (PAP: peroxidase anti-peroxidase) but
I can find no examples of its use in the existing fish gill literature. When
using HRP, endogenous peroxidases commonly associated with
erythrocytes and macrophages should be eliminated by pre-incubating the
sections with H2O2.
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Fish Osmoregulation
Immunogold labeling is generally used for determination of cellular

distribution at the subcellular level with electron microscopy although
DAB staining can also be used for this purpose. In the case of immunogold
a colloid gold particle of known size is conjugated to either the primary or
secondary antibody. Both the gold particle and DAB product are electron
dense and thus readily detectable with electron microscopy.
Immunoelectron microscopy has been used to localize Na+/K+-ATPase to
the tubular system of chloride cells (Dang et al., 2000; Wilson et al., 2000b)
and V-ATPase to pavement cells and MRCs (Sullivan et al., 1996; Wilson
et al., 2000a; Katoh et al., 2002). Silver enhancement which enables
detection of the gold particles at the light microscopic level is a very
sensitive technique but is not commonly employed (Wilson et al., 2002a;
Tresguerres et al., 2006). During the enhancement reaction silver
preferentially builds up on the surface of the gold particle until it becomes
visible with a light microscope.
CONTROLS
Controls should always be run when testing a new antibody or a new
species in order to evaluate the specificity of the staining observed.
Generally, the more controls you can run the more confidence you will
have in the specificity of your staining. However, in many cases, the best
control is not always available especially in the case of antibodies obtained
from outside the lab (commercial or by donation).
For polyclonal antibodies, the pre-immune serum (serum collected
from the same animal before immunization) serves as a good control
although normal or non-immune serum from the same species is
acceptable. Pre-immune and non-immune sera contain the normal
antibody complement that may also cross react with your tissue and can
account for non-specific labeling that you observe. An irrelevant antibody
from the same host species (and isotype) can also be used since it will be
present in super-abundant amounts similar to your own antibody which
may result in false positive labeling. Monoclonal antibody controls should
be source matched (i.e., ascites fluid, culture supernatant, or purified
immunoglobulin). In the case purified antibodies are used, then a control
antibody of the same isotype should be used (e.g., IgG2). Non-specific
labelling of the secondary antibody can be assessed by substituting the
primary antibody with dilution buffer in the labelling protocol (null
control). If an enzyme conjugate is used then endogenous enzyme activity
should be assessed by incubating the section with substrate. It is also
381
standard practice to pretreat sections with hydrogen peroxide or

lavaminsol to inhibit endogenous peroxidases and alkaline phosphatases,
respectively (see Petrusz et al., 1976; Hutson et al., 1979; Harlow and Lane,
1999)
If the antigen is available, then a blocking experiments (preincubation of the antibody with the antigen: peptide or protein) can also
be conducted. This should eliminate specific staining as the specific
antibodies will have bound to antigens in solution rather than on the
section.
In addition, when using amplification steps for immunodetection
involving biotin-avidin or streptavidin, endogenous biotin must be taken
into account in the interpretation of results. Biotin is a cofactor that is
associated with the mitochondrial enzyme pyruvate carboxylase. Thus,
cells with high mitochondrial densities would be expected to have more
abundant biotin. This biotin artifact should be apparent on negative
control slides and can be blocked if necessary (Miller et al., 2004). Heatbased antigen retrieval techniques are also capable of unmasking
endogenous biotin (Rodriguez-Soto et al., 1997).
Finally, running Western blots is advisable as the dominant
immunoreactive product can be confirmed from its predicted size. Extra
bands may be observed and the band at the predicted size may even be
absent which would be important in interpreting the ICC results. Factors
such as purity of the preparation will influence how clean the blot is since
membrane proteins only represent a small proportion of cellular proteins.
However, it is not always possible to have an antibody that is cross reactive
in both formats because the presentation of the antigen is different. An
example is the CFTR antibody 24-1 that works very well in tissue sections,
but is more difficult to get to work in Western blots.
One rather disconcerting observation that I have made is that some
antibodies cross-react with mucus either covering the gills or within goblet
cell mucin granules (Wilson et al., 2000a). In some cases, this distribution
is supported by other observations such as in the case of carbonic
anhydrase (Rahim et al., 1988). However, there other proteins in which
there is no logical reason for them to be localized to mucin granules. One
possible explanation in the case of anti-peptide antibodies is that mucin
immunoreactivity results from anti-carrier antibodies which are also
produced in the immune response. Peptides are usually too small to elicite
an immune response so they are conjugated to immunogenic carrier
molecules such as KLH or BSA. Keyhole limpet haemocyanin (KLH) is
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Fish Osmoregulation
commonly used as a carrier for peptides since it is very immunogenic and

it is also known to cross react with anti-carbohydrate antibodies (see
Thors and Linder, 2003). In this case, affinity purification of the antipeptide antibodies is usually effective. Alternatively, glycoprotein and
glycolipid oligosaccharides are also known to present a vast array of
epitopes (Feizi, 1989) and thus may be presenting an epitope similar to the
target protein. In this case, the immunologic staining is specific but not for
the target protein.
ANTIGEN-RETRIEVAL TECHNIQUES
In clinical histopathology, specimens are routinely fixed in 10% NBF
(neutral buffered formalin) and paraffin embedded for the morphological
analysis of section for disease diagnosis. Immunohistochemistry also plays
an important part in disease diagnosis. However, this form of tissue
preparation frequently leads to the reduction or loss of tissue antigenicity
particularly with monoclonal antibodies (in comparison with cryosection
which are considered the gold standard). It is, therefore, not surprising
that a great deal of attention has been focused on antigen retrieval
techniques that can recover antigenicity. There is now a vast literature
aimed at improving, and standardizing methods and understanding the
mechanisms involved (Taylor et al., 1996; Werner et al., 1996; Shia et al.,
2001). The most commonly practiced technique is heat-induced epitope
retrieval (HIER; Shia et al., 1991) with its many variants [Heating source
(microwave, water bath, autoclave, steam), temperature, time (duration,
constant, cyclic) and buffer properties (buffer, ionic composition, pH,
metals)].
Although these techniques are commonly used in histopathology and,
to a lesser extent, in basis research, there are very few examples of its use
in the fish gill literature. In my experience, antigen retrieval has in the vast
majority of cases been ineffective in resurrecting non-cross-reactive
antibodies. However, I suspect that the fault most likely lies with the nonhomologous antibody rather than the antigen. This situation is not the
same as in its clinical applications, as the antibodies are already known to
be cross-reactive in cryosections. Having said that, there are examples in
which antigen retrieval has worked. With the HIER technique V-ATPase
A subunit labeling in the guppy was greatly enhanced (J.M. Wilson,
unpublished results). In the dogfish (Squalus acanthias), the same antibody
required prior enzymatic digestion of sections with trypsin to unmask
V-ATPase A subunit epitope(s) (Wilson et al., 1997). Enzymatic digestion,
383
unfortunately, is more difficult to standardize and control than HIER

(Taylor et al., 1996). SDS (sodium dodecyl sulfate) is a strong anion
detergent that will denature proteins. A SDS pretreatment (1% SDS/PBS
for 5 min; Brown et al., 1996, Robinson and Vandr, 2001) of sections has
been shown to improve a5 immunoreactivity (rat kidney; Sabolic et al.,
1999) and it is my general experience with fish as well. SDS pretreatment
was also necessary for apical tAE1 labeling in chloride cells in fresh water
tilapia (Wilson et al., 2000a) and coho salmon (Wilson et al., 2002b). In
both the case of a5 and tAE1, the antigen used to raise the antibodies had
been denatured at some point in their purification and SDS denaturation
may alter the respective epitopes and improve antibody binding.
Citraconic anhydride has been recently demonstrated to be a very
effective antigen retrieval agent at high temperature and physiological pH
(Naminatsu et al., 2005). Citraconic anhydride acts by reversing the
chemical cross linking by formaldehyde fixation. So far, I have found it to
be a useful method for retrieving antigenicity in a number of cases where
results were initially negative or weak. In larval lamprey, Na+/K+-ATPase
activity and protein level expression in Western blots were low but
detectable in gill tissue compared to juvenile lamprey. With a standard
immunolabeling protocol, I was unable to demonstrate immunoreacitivity
in sections but with 0.05% citraconic annhyride treatment of sections for
30 min at 98C labeling with either the a5 or aRbNKA antibodies was
dramatically improved.
In Figures 13.5 and 13.6, I have attempted to illustrate the effects of
various antigen retrieval techniques on antigen detection. In sturgeon gill,
Na+/K+-ATPase (a5 and aRbNKA), V-ATPase (B subunit) and NKCC
(T4) staining were weak under standard conditions using an indirect
labeling protocol(i) . Antigen retrieval with 1% SDS /PBS for 5 min at room
(i)
Gill tissue was fixed in 3% PFA/PBS overnight, dehydrated through a graded EtOH series,
cleared with ClearRite (Richard-Allen) and infiltrated and embedded in paraffin (Type 6,
Richard-Allen). Paraffin sections (5 mm) were collected onto APS (3aminopropyltriethoxysilane; Sigma MO) coated slides, completely air dried, and dewaxed in
Clear-Rite (Richard Allen). Sections were circled with a hydrophobic barrier (DakoPen,
Dako), and rehydrated with 5% normal goat serum in 0.1% BSA/ TPBS (0.05%Tween-20/
Phosphate Buffered Saline, pH 7.4) for 20 min. Sections were then incubated with primary
antibody diluted 1:200 1:500 in BSA/TPBS for 1 h at 37C. Slides were rinsed in TPBS (5,
10, 15 min in Coplin jars), and incubated with goat anti-mouse Alexa Fluoro 488 and goat
anti-rabbit Alexa Fluoro 594 conjugated secondary antibodies both diluted 1:200 (Molecular
Probes Inc) in BSA/TPBS for 1 h at 37C. Following a second round of rinses in TPBS,
coverslips were mounted using a glycerol based fluorescence mounting media (10% Mowiol,
40% glycerol, 0.1% DABCO, 0.1 M TRIS (pH 8.5). Sections were viewed on a Leitz Ortholux
2 epi-fluorescence microscope and images captured using a digital camera (Leica DFC300).
384
Fish Osmoregulation
Fig. 13.5 Demonstration of the effects of different antigen retrieval techniques on

immunoreactivity of sturgeon gill filament sagittal sections double labeled with antibodies
against Na+/K+-ATPase (aRbNKA) and NKCC (T4) with phase contrast images for
orientation. Section pre-treatments: (a, a,a, respectively) Control (no pre-treatment), (b,
b, b, respectively) 1% SDS/PBS pH 7.3 for 5 min at room temperature, or heat induced
epitope retrieval (HIER; (c, c, c, respectively) 10 mM Sodium Citrate pH 6 or (d, d, d,
respectively) 0.05% citraconic anhydride pH 7.4 for 30 min at 98C (J.M. Wilson, D. Baker
and C.J. Brauner, unpublished observations). Scale bar = 50 mm.
temperature improved Na+/K+-ATPase labeling with both antibodies

although background staining was much higher with the a5 antibody.
With the NKCC T4 antibody results were only slightly improved and with
the V-ATPase B2/BvA1 antibody only background staining was increased.
Heat-induced epitope retrieval (HIER) improved the immunoreactivity of
385
Fig. 13.6 Antigen retrieval of sturgeon gill sections probed with antibodies against Na+/K+ATPase (a5) and V-ATPase B subunit (B2/BvA1, unpublished). Section pre-treatments:
Section pre-treatments: (a, a,a) Control (no pretreatment), (b, b, b) 1% SDS/PBS pH
7.3 for 5 min at room temperature, or heat induced epitope retrieval (HIER; (c, c, c) 10
mM Sodium Citrate pH 6 or (d, d, d) 0.05% citraconic anhydride pH 7.4 for 30 min at 98C
(J.M. Wilson, D. Baker and C.J. Brauner, unpublished observations). Scale bar = 50 mm.
all antibodies used in this example. Heating the slides in either 10 mM

sodium citrate (pH 6) or 0.05% citraconic anhydride at pH 7.4 for 30 min
at 98C using a conventional heat source (hotplate) gave similar results for
the most part. Generally, the staining was more consistent throughout the
sections. The citraconic anhydride treatment did however give superior
results with the T4 antibody.
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Fish Osmoregulation
Thus, despite the lack of attention so far given to antigen retrieval

techniques, they stand to provide a second life to some antibodies long
thought useless. Since, signal strength is generally increased with these
methods it is essential that negative controls are run in parallel in order to
properly evaluate the results.
ADDITIONAL RESOURCES
As this review is far from complete, the reader is urged to acquire Lane and
Harlows (2000) book Using Antibodies for more substantial explanations
and detailed protocols. An excellent online discussion group for asking
questions is Histonet and the archives are searchable (Histosearch). For
those interested in testing primarily non-homologous antibodies,the
following web-based search engines are useful for finding appropriate
commercial antibody: www.abcam.com and www.exactantigen.com. The
monoclonal antibodies available through the Developmental Studies
Hybridoma Bank can be found at www.uiowa.edu/~dshbwww.
Acknowledgements
The published and unpublished data of JMW included in the review were
made possible by the Portuguese Foundation for Science and Technology
(FCT) grants POCTI/ BSE/ 34164/ 2000, POCTI/ BSE/ 47585/ 2002 and
POCTI/ MAR/ 60365/ 2004. JMW was supported by an FCT auxiliary
investigator position to CIMAR.
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+0)26-4
14
Rapid Regulation of Ion Transport
in Mitochondrion-rich Cells
William S. Marshall
INTRODUCTION
Mitochondrion-rich Chloride Secreting Cells
Mitochondrion-rich (MR) cells are present in large numbers in the gill and
opercular epithelia of estuarine teleosts. The cell density is augmented in
hypersaline conditions and moderates slightly in brackish water, but in the
wild these epithelia are capable of rapid chloride secretion. Typically, the
cells appear with a smaller accessory cell entwined with the larger cell and
between the accessory cells and MR cells is a leaky paracellular pathway
(Sardet et al., 1979) that allows transepithelial transport of sodium down
its electrochemical gradient (Degnan and Zadunaisky, 1980). The
opercular membrane of killifish (Fundulus heteroclitus) (Degnan et al.,
1977; Karnaky and Kinter, 1977; Degnan and Zadunaisky, 1980), of tilapia
Authors address: Department of Biology, St. Francis Xavier University, P.O. Box 5000,
Antigonish, Nova Scotia, Canada B2G 2W5.
E-mail: bmarshal@stfx.ca
396
Fish Osmoregulation
(Oreochromis mossambicus) and the skin of the euryhaline goby (Gillichthys

mirabilis) (Marshall, 1977; Marshall and Bern, 1980) provide convenient
flat preparations of gill-like epithelia that actively secrete Cl at high rates.
The marine MR cell model (Marshall and Bryson, 1998; Evans et al., 1999,
2005; Marshall, 2002; Marshall et al., 2002; Fig. 14.1) includes a
basolateral Na+,K+,2Cl symport (NKCC) or cotransporter of the NKCC1
type that transports Cl into the cytosol, driven by the transmembrane
Na+ gradient. The Na+ gradient is maintained by Na+,K+-ATPase that is
located on the tubular system that is made up of infoldings of the
Fig. 14.1 Model of the seawater (SW) type mitochondria-rich cell complex with Na+,K+ATPase, NKCC1 and K+ channels on the basolateral membrane that accumulate Cl in the
cytosol driven indirectly by the pump. Solid arrows and closed symbols indicate active
transport; dashed lines and open symbols, passive processes. Cl exits through CFTR like
anion channels in the apical membrane that has a typical cup shaped apical crypt.
Intercellular junctions between mitochondria rich (MR) cells and pavement cells (P) are
many stranded and have low permeability, while junctions between accessory cells (AC)
and MR cells are single stranded and provide a high conductance paracellular pathway that
is cation (Na+) selective. Also shown is the proposed uptake pathway for transepithelial
Ca2+ uptake, via Ca2+ channels at the apical membrane and via Na/Ca2+ and Ca2+-ATPase
at the basolateral membrane. Acid/base and other solute transport pathways are not
shown.
William S. Marshall
397
basolateral membrane. Cl secretion can be blocked by basolateral

furosemide and bumetanide, loop diuretics that block NKCC type
cotransporters, but not by thiazide type diuretics that block Na+-Cl
symports (Marshall and Bryson, 1998). Intracellularly, the Cl diffuses to
the apical membrane where anion channels conduct Cl across the
epithelium and into the environment. The anion channel in the apical
membrane is a homolog of Cystic Fibrosis Transmembrane Conductance
Regulator (CFTR), having pharmacological and single channel properties
similar to h<FTR (Marshall et al., 1995) and cloned CFTR from killifish,
when expressed in amphibian oocytes, imparts a cAMP stimulated anion
conductance (Singer et al., 1998). Cl exit across the apical membrane
generates a blood side-positive electrical potential that is sufficient to
drive Na+ out via a cation-selective paracellular pathway. Epithelial
current and ion conductance are localized to MR cells, measured using the
vibrating microprobe (Foskett and Scheffey, 1982). The anion channel has
a low conductance, 8 pS, and deactivates on excision of an inside out
membrane patch. Addition of Protein Kinase A (PKA) and ATP activates
the channels in quiescent patches, demonstrating that phosphorylation
activates the channel. The Cl transepithelial current is sensitive to the
anion channel inhibitors diphenyl amine-2-carboxylate (DPC) and 5nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) but insensitive to
4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS; Marshall et al.,
1995; Marshall and Bryson, 1998). The channel has been cloned,
sequenced and found to be homologous to hCFTR (Singer et al., 1998).
Further, the expressed gene in Xenopus oocytes also produces a cAMP
activated anion current (Singer et al., 1998).
Since NKCC1 also carries K+ into the cell, K+ must be recycled across
the basolateral membrane and transepithelial Cl secretion thus is blocked
by K+-free solutions and by barium. The K+ channel in the basolateral
membrane of gill epithelial cells has tentatively been identified (Duranton
et al., 2000a, b) and found to be a large conductance (122 pS) channel
activated by hypotonicity and by ionomycin, while being inhibited by the
addition of quinidine and charybdotoxin. There appear to be separate
basolateral and apical membrane K+ channel populations (Duranton et al.,
2000a).
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Fish Osmoregulation
CONTROL OF Cl SECRETION
Overview
The hormonal control of osmoregulation in fish has been recently
reviewed. McCormick (2001) and Evans (2002) specifically studied cell
signalling in fish gills. Many factors are known to affect chloride secretion
in teleost fish pharmacologically (Table 14.1), including hormones and
neurotransmitters that inhibit secretion: catecholamines, via a-2
adrenoceptors, acetylcholine via muscarinic receptors, somatostatin and
urotensin II. The second messenger pathway for adrenergic and
cholinergic receptors appears to be calcium. From mammalian research on
urotensin II action, there is also an association with intracellular calcium
(Conlon et al., 1997). There are also many hormones, neurotransmitters
and drugs that stimulate chloride secretion: vasoactive intestinal
polypeptide (VIP), glucagon, arginine vasotocin (AVT) and urotensin I
(Table 14.1) and in these cases the common pathway appears to be an
augmentation of intracellular cAMP, activation of PKA. For instance, the
b-adrenergic agonist isoproterenol at 1.0 mM causes rapid accumulation of
cAMP in killifish opercular epithelia.
Beyond the second messengers, the termination of the
phosphorylation and dephosphorylation cascades are of interest, as
cotransporters and channels are now known to have tightly associated
kinases and phosphatases including complexes involving scaffolding
proteins. In spite of the rapid action, there exists a complex series of steps
that follow the second messenger changes and provide the unique
regulation of these ion transport systems. Elucidation of the regulatory
pathways terminating at channels and transporters will be important to
understanding ion transport regulation in health and disease.
Catecholamines
=-adrenoceptors
The most likely physiologically relevant response is the inhibition of Cl
secretion by epinephrine and norepinephrine. The response is rapid, dosedependent and occurs at catecholamine levels that are physiologically
realistic, with ED50 of approximately 50 nM for epinephrine and 500 nM
for norepinephrine. The effect is blocked by yohimbine, indicating an
a-receptor type (May et al., 1984; Marshall et al., 1993; Marshall and
William S. Marshall
399
Table 14.1 Summary of potential rapid secretagogues and inhibitory agents affecting Cl
secretion by marine teleost mitochondrion-rich (MR) cells.
Agent added
Effect on Cl 2nd Messenger

secretion
Hormones
Arginine vasotocin
cAMP
Glucagon
Urotensin I
Atrial natriuretic peptide
0?
cAMP
cAMP
0
Epinephrine
Ca2+
Urotensin II
Ca2+
Neurotransmitters and Parahormones

Vasoactive intestinal peptide
Serotonin
cAMP
0
Norepinephrine
Ca2+
Acetylcholine
Ca2+
Endothelin
NO (sodium nitroprusside)
Prostaglandin (PGE2)
Somatostatin
?
?
?
cAMP
Agents
Calyculin A
IBMX/db-cAMP
cAMP
A23187
ionomycin
Furosemide, Bumetanide
Ca2+
-
DPC, NPPB (Apical)
Glibenclamide
Genistein
References
Avella et al. (1999)

Marshall (2002)
Foskett et al. (1982)
Marshall and Bern (1980)
Scheide and Zadunaisky
(1988)
Evans (2002)
Degnan et al. (1977)
Marshall and Bern (1980)
Avella et al. (1999)
Marshall and Bern
(1979, 1980)
Marshall et al. (1993)
Evans (2002)
May et al. (1984)
May and Degnan (1985)
Evans (2002)
Evans (2002)
van Praag et al. (1987)
Davis and Shuttleworth
(1986)
Hoffmann et al. (2002)
Mendelsohn et al. (1981)
May et al. (1984)
May and Degnan (1985)
Marshall et al. (1993, 2000)
Eriksson et al. (1985)
(Table 14.1 contd.)
400
Fish Osmoregulation
(Table 14.1 contd.)
Treatments
Hypertonic shock
Hypotonic shock
Ca2+?
Zadunaisky et al. (1995)

Marshall et al. (2000, 2005)
Daborn et al. (2001)
Leguen and Prunet (2004)
+: stimulation, -: inhibition, 0: No effect.
Bryson, 1998). The a2-receptor subtype was determined by comparing

affinity for norepinephrine and epinephrine (Marshall et al., 1993) by
application of specific a2-receptor agonist clonidine (May et al., 1984;
Marshall et al., 1993), by comparing a1-adrenergic agonist phenylephrine
with clonidine (Marshall et al., 1993) and by use of a2-receptor blockers
to inhibit epinephrine action (May et al., 1984).
Physiological inhibition is via =2 -adrenoceptors
However marked the effect of b-adrenoceptor agonist isoproterenol might
be in stimulating chloride secretion (see below), the application of the
natural non-specific agonists (epinephrine and norepinephrine) invariably
causes strong inhibition of the chloride secretion in tilapia opercular
membranes (Foskett et al., 1982; Foskett and Machen 1985), Fundulus
opercular membranes (Degnan et al., 1977; May and Degnan, 1985;
Marshall et al., 1993) and goby (Gillichthys mirabilis) skin (Marshall and
Bern, 1980). The b-adrenergic response, therefore, appears to be
pharmacological. The ED50 for epinephrine is between 0.1 and 1.0 mM for
opercular membrane of tilapia (Foskett et al., 1982) and Fundulus
(Marshall et al., 1993) and Gillichthys skin (Marshall and Bern, 1980).
Circulating catecholamine levels in resting, unstressed teleosts are below
this range (0.05 mM); thus, Cl secretion is not likely under tonic
inhibition by the interrenal. In stressed teleosts, catecholamine levels are
much higher (0.5 1.0 mM) and would inhibit Cl secretion by the gills.
Spontaneous recovery of Cl secretion after epinephrine addition is
observed in killifish and tilapia membranes and was initially ascribed to
b-adrenergic activity, but was prevented by addition of antioxidants
(ascorbate) (Foskett et al., 1982), so oxidation of the catecholamine would
appear to be the cause of the observed recovery.
The opercular epithelium was dissected with its nerve supply intact,
mounted in an Ussing chamber and the nerve directly stimulated while
William S. Marshall
401
measuring transepithelial Cl secretion (Marshall et al., 1998). Stimulation

of the branch of the glossopharyngeal nerve that innervates the opercular
epithelium in isolated epithelia illustrates that the natural sympathetic
reflex response in the opercular epithelium (and likely also the gill MR
cells) is inhibition of chloride secretion. Autonomic nerves track alongside
cranial nerves leading to the gill and head region of teleosts (Sundin and
Nilsson, 2002), so stimulation of the bundle would activate autonomic and
motor neurons. Nerve stimulation induced rapid inhibition that was
unaffected by cholinergic blockers (atropine and tubocurarine) but was
blocked effectively by the a-adrenergic inhibitor yohimbine (Marshall
et al., 1998), demonstrating a-adrenergic mediation. Thus, physiological
activation of autonomic neurons innervating the opercular epithelium and
gill produce rapid inhibition of Cl secretion.
Catecholamine effects on the gill vasculature are consistent with the
above-mentioned inhibition of epithelial Cl secretion. Epinephrine
induces a reduction in perfusion of the ion transporting cells, in that
epinephrine dilates the arterio-arterial perfusion of flounder gill lamellae
at the expense of the arterio-venous flow that supplies the gill filament
epithelium where the MR cells reside (Stagg and Shuttleworth, 1984). In
rainbow trout (Oncorhynchus mykiss), epinephrine increases functional
surface area for gas exchange, a combination of vasodilation via
b-receptors on the afferent side and a balanced vasoconstriction on the
efferent side mediated by a-receptors, reviewed by Sundin and Nilsson
(2002). These changes are consistent with a shift away from perfusion of
ion transporting cells more toward perfusion of the lamellae.
Calcium as second messenger
In many epithelial transport systems, intracellular calcium is associated
with stimulation of transport. This is true for avian salt gland, reptilian salt
gland, mammalian airway epithelium (Liedtke, 1990), mammalian sweat
gland and elasmobranch rectal gland (Evans, 2002). Thus, teleost MR
cells are somewhat exceptional in having a calcium-mediated inhibition of
NaCl secretion. Calcium is apparently the second messenger for many
inhibitory hormones and neurotransmitters in gill MR cells (Table 14.1)
and may also be involved in hypotonic shock (Leguen and Prunet, 2004;
also see below).
402
Fish Osmoregulation
>-adrenoceptors are also present

Isoproterenol stimulates Cl secretion in killifish opercular epithelium
(May et al., 1984; Marshall et al., 1998) and Gillichthys skin (Marshall and
Bern, 1980). While specific b1 and b2 agonists were ineffective and thus
did not identify the b receptor subtype, b1 antagonists (e.g., timolol)
blocked the response to isoproterenol, whereas the b2 antagonists were
ineffective, indicating the presence of a b1 receptor subtype (May and
Degnan, 1985). The b-adrenoceptor second messenger is cAMP, as
isoproterenol (Stagg and Shuttleworth, 1984; Marshall et al., 2000) and
norepinephrine (Stagg and Shuttleworth, 1984) significantly increase
opercular epithelium tissue content of cAMP. It has been suggested that
the b-adrenergic response might enhance recovery from an initial
a-adrenergic inhibition.
Acetylcholine, Serotonin, Dopamine
Acetylcholine decreases Cl secretion rate in killifish opercular
membranes via muscarinic receptors, based on blockade of the effect by
homatropine (Mendelsohn et al., 1981). The response is not mediated by
cAMP decrease, as there was no change in cAMP levels after
acetylcholine treatment, neither did acetylcholine decrease cAMP levels
that had been stimulated by isoproterenol (May and Degnan, 1985).
Because the a-adrenergic and cholinergic responses mirrored each other
in a variety of pharmacological treatments, May and Degnan (1985)
proposed that the two responses converged on a common inhibitory
pathway not involving cAMP lowering.
Serotonin is known to stimulate epithelial Cl secretion in dog
tracheal epithelium (Tamaoki et al., 1997) and rabbit corneal epithelium
(Marshall and Klyce, 1984) and serotoninergic neurons have been
identified in teleost gills (Bailly et al., 1992; Sundin and Nilsson, 2002)
particularly in proximity to smooth muscle. However, serotonin at low
doses (1 and 10 mM) added to the killifish opercular epithelium has no
effect; at high doses (50 mM) it is modestly inhibitory and appears to be
acting through adrenergic receptors because the effect is blocked by
yohimbine (Marshall et al., 1993). Dopamine has not been tested in the
opercular membrane system to see if this substance could potentially
regulate Cl secretion.
William S. Marshall
403
Urotensin I and Urotensin II

Urotensin I is a peptide resembling mammalian Corticotrophin Releasing
Factor (CRF) and amphibian sauvagine that is secreted by the caudal
neurosecretory system of teleosts (Bern et al., 1985; Pohl et al., 2001).
Urotensin I stimulates Cl secretion in Gillichthys skin MR cells (Marshall
and Bern, 1979, 1981) and the effect is more apparent in epithelia that
were previously inhibited by epinephrine. However, thus far, urotensin I
receptors have not been localized in the gills of teleosts, while three
different CRF type receptors have been localized in catfish to brain, heart,
pituitary and urophysis (Arai et al., 2001). The potent vasodilatory effects
of urotensin I would suggest primarily a role in these responses (Olson,
2002).
Urotensin II is a Somatostatin-like peptide that is secreted by the
caudal neurosecretory system of teleost fish (Bern et al., 1985). Urotensin
II is most prevalent in the caudal neurosecretory system (Bern et al., 1985)
but has now been identified in the brain of many vertebrates, suggesting
an ancient neurosecretory role in vertebrates (Conlon et al., 1997).
Urotensin II significantly inhibits Cl secretion by the goby skin (Marshall
and Bern, 1979, 1981) and tilapia opercular membrane (Foskett and
Hubbard 1981; Foskett et al., 1982), an effect reversed by agents that
augment tissue cAMP. Plasma levels measured by homologous RIA
indicate that seawater (SW) adapted flounder (Platichthys flesus) had
higher plasma urotensin II than their freshwater counterparts (Winter
et al., 1999). While the urotensins are associated with ion balance in fish,
their release into the renal portal system by neurosecretory cells of the
urophysis points toward a renal and intestinal responses (Bern et al., 1985)
rather than a more systemic effect on more remote osmoregulatory
structures such as the gill and opercular membranes. Urotensin II levels in
plasma of flounder transferred from SW to FW were significantly lower
than SW-SW controls at 24 and 72 h after transfer (Bond et al., 2002),
suggesting downregulation of urotensin II during FW acclimation. In both
transfer groups, the disturbance appeared to inhibit urotensin II release
compared to long term acclimated FW or SW animals that had 2025 fmol/ml urotensin II (Bond et al., 2002).
VIP, Glucagon and Atrial Natriuretic Peptide
Vasoactive Intestinal Polypeptide (VIP) rapidly increases Cl secretion by
the tilapia opercular epithelium if the Cl secretion had been previously
404
Fish Osmoregulation
inhibited by epinephrine (Foskett et al., 1982), while VIP alone applied to

uninhibited epithelia had only minimal response. The response is
consistent with a cAMP mediated effect because addition of small
amounts of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine
augmented the VIP response. Similar results to VIP were obtained by
Foskett et al. (1982) using glucagon. In both cases, the doses necessary to
produce marked stimulation of the Cl secretion were rather high
(105 M). VIP and glucagon at physiological concentrations (1013 1011 M) stimulate adenylate cyclase in SW acclimated rainbow trout gill
membranes, more so than in freshwater trout (Guibbolini and Lahlou,
1987, 1990). VIP (26-28 amino acids) belongs to the glucagon like
peptides and teleost VIP shares high similarity to human VIP (85 and 88%
at the protein level for Takifugu rubripes, fugu and Danio rerio, zebrafish,
respectively), while zebrafish and fugu VIP share 77% amino acid
similarity. However, the SW teleosts (fugu and Atlantic cod, Gadus
morhua) have a phenylalanine in position 13 rather than leucine
(Mammal) and variations in the amino terminal amino acids (Table 14.2).
These variations may explain the apparent insensitivity of the tilapia
receptors for mammalian VIP (Foskett et al., 1982). The variation in VIP
sequence suggests that human VIP could activate teleost VIP receptors
but that glucagon could also be acting through the VIP receptors. VIP
receptors have been identified in gill and other tissues of goldfish,
Carassius auratus (Chow et al., 1997) and the EC50 for cod VIP on goldfish
VIP receptor is 1 nM but that the receptor will also bind glucagon at a
lower affinity (Chow et al., 1997). The high doses of heterologous glucagon
and VIP required in earlier work suggest that native VIP will be needed to
examine truly physiological responses. In nature, VIP responses would
most likely be mediated by VIP neurons rather than by circulating VIP. In
goldfish, there exist VIP immunoreactive cells on the gills (Chow et al.,
1997) and in the goldfish gill there are immunoreactive VIP receptors in
nerve cell bodies in the subepithelial space (de Girolamo et al., 1997). The
position of these VIP neurons in the gill suggests a possible physiological
response to VIP. VIP is vasodilatory in brown trout (Salmo trutta) gill, an
effect blocked by indomethacin, hence suggesting a vasoactive response
mediated by prostaglandins (Bolis et al., 1984). While it is not known
specifically that neurally derived VIP stimulates Cl secretion by the gill,
these results collectively point toward such a response.
Atriopeptin II (109-107 M), an Atrial Natriuretic Peptide (ANP), is
reported to have a modest stimulatory effect on Cl secretion by the
William S. Marshall
Table 14.2
405
Transporter regulation.
Transporter
Apical Membrane
CFTR Anion Channel
Basolateral membrane
NKCC1 cotransporter
NKCC1 cotransporter
Stimulus
(Second messenger)
Downstream
events
Reference
(cAMP)
PKA
FAK
Marshall et al. (1995, 2005a)

Marshall et al. (2005b)
Hypotonicity
(Ca2+)
p38 MAPK
JNK
Hypertonicity
ERK1
SRC?
pY407 FAK
PP1?
PKC
Marshall et al. (2005a)

Kltz and Avila (2001)
p38 MAPK
JNK
NKCC1 cotransporter
Na +,K +-ATPase
K+-channel
cAMP
?
?
MLCK
pY407 FAK?
OSR1/SPAK?
PKA?
?
?

: activation/phosphorylation, deactivation/dephosphorylation
PK protein kinase, ERK1 extracellular signal regulated protein kinase, MAPK mitogen-activated protein kinase,
JNK J-N-terminal kinase, FAK focal adhesion kinase (pY407 , phosphorylation at tyrosine position 407), MLCK
myosin light chain kinase, OSR1 oxidative stress response protein kinase, SPAK ste20-like proline-rich protein
kinase, PP protein phosphatase.
killifish opercular epithelium from SW and freshwater adapted animals

(Scheide and Zadunaisky, 1988). The effect was not mediated by neural
activity, as the effect was not blocked by tetrodotoxin; likewise the effect
was not mediated by adrenergic receptors, as blockade of b adrenoceptors
with propranalol did not affect the response. ANP receptors in European
eel (Anguilla anguilla) gills activate cGMP pathways and in SW eels
branchial cells more than in freshwater branchial cells (Broadhead et al.,
1992). Eel gills rapidly remove ANP from circulation and ANP and CNP
have clear effects on drinking rates and intestinal salt transport in eels
especially during the early phase of SW adaptation (Takei and Hirose,
2002). Eel ANP, eel CNP and the NPR-C-specific C-ANF inhibited the
406
Fish Osmoregulation
forskolin-stimulated production of cyclic AMP in dispersed eel gill cells

(Callahan et al., 2004) and this effect was abolished by pre-treatment of
cells with pertussis toxin. Whereas ANP likely has some function in teleost
gill cells, the re-examination of ANP for an ion transport effect has failed
to confirm the ANP response in this system (Evans, 2002).
Arginine Vasotocin
There is a detailed case for arginine vasotocin (AVT) regulation of Cl
secretion and related to SW adaptation. AVT specific binding to eel gill
cells is higher in SW adapted animals than FW eels (Guibbolini et al.,
1988), strong evidence for the existence of AVT receptors and a function
in SW osmoregulation. The AVT receptor has been isolated from flounder
gill and from white sucker, Catostomous commersoni (Mahlmann et al.,
1994; Pierson et al., 1995; Warne 2001; Genbank AF184966). From
Northern analysis, the AVT receptor is expressed in gill as well as brain,
kidney and liver. Expression of AVT receptors in Xenopus oocytes results
in dose-response stimulation of an inward current, likely anion current,
using native AVT and the mammalian V1 AVT agonist. In rainbow trout,
there appears to be an increase in plasma AVT when the animals are
moved to higher salinity (Pierson et al., 1995). Measurement of plasma
AVT in flounder transferred from SW to FW revealed marked inhibition
of AVT release compared to control fish that were transferred from SW to
SW (Bond et al., 2002), indicating a possible role for AVT in SW. More
directly, AVT plasma levels are correlated with plasma tonicity and rise
significantly in flounder infused with NaCl (Warne and Balment, 1995),
consistent with AVT release being stimulated by increases in plasma
osmolality.
Whereas AVT has not been shown to stimulate Cl secretion by the
opercular epithelium, the cultured gill cell epithelium from sea bass
(Dicentrarchus labrax) shows a modest increase in Cl secretion with 50 nM
AVT (Avella et al., 1999). Although the demonstrated effective AVT dose
in vitro is much higher than measured plasma levels in vivo, it remains a
possible upregulator that could restore SW level ion secretion. While
glucagon increases adenylate cyclase activity in trout gills, AVT and
isotocin are inhibitory (Guibbolini and Lahlou, 1992).
William S. Marshall
407
Nitric Oxide and Endothelin

The recent observation that nitric oxide synthase co-localizes with
mitochondria rich cells in killifish opercular epithelium (Evans, 2002)
invites consideration of this effector in regulation of Cl secretion. NO
donors such as sodium nitroprusside produce a dose-dependent inhibit Cl;
secretion by killifish opercular membranes. NO donors also inhibit
Na+,K+-ATPase in gill homogenates from brown trout (Towle et al., 1977;
Tipsmark and Madsen, 2003). In Atlantic salmon (Salmo salar) smolts,
NOS colocalizes with Na+,K+-ATPase in gill MR cells and there is an
increase in NADPHD in the gill during smolting suggests a regulatory role
of NO in the attenuation of the smolting-related increase in Na+,K+ATPase activity prior to entering SW (Ebbesson et al., 2005). It is possible
that other agents may be acting through NO, here potentially acting in a
paracrine/autocrine manner.
Big endothelin immunoreactivity is identified in mitochondria rich
cells of Atlantic stingray (Dasyatis sabina), suggesting presence of the
peptide in gill ion transporting cells (Evans, 2002). Addition of endothelin
agonists to killifish opercular epithelia shows a concentration dependent
inhibition of Cl secretion with a threshold of 1010 M (Evans, 2002).
There is growing evidence that NO and endothelin may interact to
downregulate Cl secretion.
Prostaglandins and Eicosanoids
The opercular epithelium and gills of killifish metabolize eicosanoids and
produce prostaglandins and leukotrienes (van Praag et al., 1987). PGE2
rapidly inhibits Cl secretion by the opercular epithelium (Eriksson et al.,
1985; van Praag et al., 1987), whereas several leukotrienes appear to
stimulate Cl secretion (van Praag et al., 1987). In cultured respiratory
cells from sea bass gills, PGE2 has a small stimulatory effect, apparently on
Cl secretion (Avella et al., 1999). In gill filaments and kidneys of both eel
and trout, PGE2 is present but PGD2 and 6-keto PGF1a were major
prostaglandins and concentrations of these were significantly lower in the
eel after SW adaptation (Brown et al., 1991). Rainbow trout gill filaments
synthesize a wide range of eicosanoid products following calcium
ionophore challenge, indicating potential functions in freshwater gill
function (Holland et al., 1999). Prostaglandins are synthesized via COX2, as the specific COX 2 inhibitor NS-398 inhibitor blocks the endothelin
408
Fish Osmoregulation
response (Evans et al., 1999). PGF2a, PGD2, PGI2 and thromoboxane A2

are all ineffective (Evans et al., 1999). As has been mentioned above,
prostaglandins apparently mediate the VIP responses in gill vasculature,
but thus far a prostaglandin that stimulates Cl secretion has not been
identified, although such may exist because application of arachidonic acid
alone produces a biphasic response where the first phase is stimulatory
(van Praag et al., 1987).
Mediation of Cl secretion stimulation
One locus for cAMP mediated stimulation of Cl secretion appears to be
an apically located CFTR-like anion channel. The channel was described
in patch clamp experiments on killifish MR cells to be a low conductance
(8 pS) anion selective channel that is activated by cAMP (Marshall et al.,
1995). The killifish CFTR gene, cloned and expressed in amphibian
oocytes imparts a cAMP activated current (Singer et al., 1998). In excized
patches from MR cells, protein kinase A and ATP addition activate
CFTR-like channels in quiescent patches (Marshall and Singer, 2002).
The b adrenergic agonist isoproterenol (May and Degnan, 1985; Marshall
et al., 2000) and forskolin (May and Degnan, 1985) rapidly increase
killifish opercular membrane cAMP levels accompanied by large increases
in epithelial short-circuit current. It is thus clear that cAMP activates the
apical membrane chloride conductance and transepithelial chloride
secretion.
It is still possible for participation of NKCC activation at the
basolateral membrane as well, but this has not been demonstrated in
teleost preparations. Indeed NKCC phosphorylation is a common yet
complex regulatory mechanism in NKCC containing systems (Flatman,
2002) and full activation of transepithelial transport would require
activation of both components in a coordinated fashion.
In the dogfish shark (Squalas acanthias) rectal gland, that secretes Cl
in a manner similar to teleost MR cells, isoproterenol activates secretion
via cAMP and increases phosphorylation of NKCC threonine residues in
the N terminus (Flemmer et al., 2002). This mode of NKCC activation
seems widely distributed, as similar responses were observed in at least two
other NKCC secretory systems: rat parotid gland and tracheal epithelium
(Flatman, 2002). The protein serine/threonine phosphatase (PP) PP1 and
PP2A inhibitor calyculin A stimulates NKCC1 in several systems
(Flatman, 2002) and stimulates Cl secretion by the killifish opercular
William S. Marshall
409
membrane (Hoffmann et al., 2002), hence the model includes PP1 and
PP2A as possible downregulators of NKCC1 and activation of NKCC1 via
cAMP and PKA.
Short-term activation of Na+,K+-ATPase and basolateral K+
channels, could feasibly also contribute to upregulation of Cl secretion
through enhancement of the Na+ transmembrane gradient that drives
NKCC1. Rapid activation of Na+,K+-ATPase, within hours of transfer to
SW, was observed in killifish gill (Towle et al., 1977; Mancera and
McCormick, 2000). Short-term activation of the enzyme in killifish gills
can be as rapid 0.5 h (Towle et al., 1977) to 3.0 h (Mancera and
McCormick, 2000) after transfer to SW and the response is dependent on
protein synthesis (Mancera and McCormick, 2000). Upregulation of K+
channels, in turn could also increase turnover rate of Na+,K+-ATPase by
enhancement of K+ recycling across the basolateral membrane and
supplying NKCC with K+ extracellularly. Blockade of K+ channels by
barium (Degnan, 1985; Zadunaisky et al., 1995) rapidly inhibits Cl
secretion by the opercular epithelium, demonstrating the dependence on
operational basolateral K+ conductance.
Mediation of Cl transport inhibition
Much less is known regarding the downregulation of Cl secretion and the
subsequent covering of inactive Cl cells by pavement cells. The calcium
ionophore ionomycin reduced Cl secretion with a similar time course to
that of clonidine (Marshall et al., 1993) but the less efficient calcium
ionophore A23187 is ineffective (Degnan et al., 1977; Stagg and
Shuttleworth, 1984; Marshall et al., 1993), presumably because in some
poikilothermic systems A23187 is not sufficiently active. The second
messenger for the a2-adrenergic response appears to be calcium, based on
a blunting of the clonidine response in calcium (Marshall et al., 1993)
depleted media and the fact that clonidine did not decrease tissue levels
of cAMP in killifish opercular epithelium (Marshall et al., 1993). Whereas
calcium activation of Cl secretion is the norm for many otherwise similar
systems from diverse origins, including dogfish shark rectal gland, colon,
airway and avian salt gland, the effect of calcium in teleost MR cells is
inhibitory. The source of this regulatory inversion is assumed to be a
terminal phosphatase instead of a kinase in a calcium-regulated regulatory
pathway.
410
Fish Osmoregulation
Fig. 14.2 Hypothetical model of apical membrane transport in rapid control of Cl

secretion by seawater type mitochondria rich cells of marine and estuarine teleosts. Apical
membrane regulation of CFTR anion channels includes cAMP activation via protein kinase
A (PKA) and phosphorylation of the channel. Inferred from mammalian results is the
involvement of EZRIN, RADIXIN, MOESIN linkage to the PDZ domain at the carboxy
terminus of CFTR and the downregulation of CFTR by syntaxin 1a and SNAP/SNARE
proteins.
HYPOTONIC AND HYPERTONIC STRESS AND Cl

SECRETION
Hypotonic Shock
Hypotonic shock and cell swelling inhibits Cl secretion by the killifish
opercular epithelium in a dose-dependent fashion. The reduction in Cl
secretion was approximately 60% for a 40 mOsm/kg reduction in
basolateral osmolality (Marshall et al., 2000). The effect is freely reversible
by reintroduction of iso-osmotic solutions, usually with an overshoot.
Reduction in NaCl with added mannitol to maintain constant osmolality
was a control that had no effect on Cl secretion rate, therefore the
hypotonic effect is purely osmotic. Hypotonic shock on the basolateral side
produced the same effect as bilateral reductions in osmolality. Hypotonic
shock had no effect on tissue cAMP levels and was not impeded by pretreatment of the tissue with the Ca2+ ionophore ionomycin or the calcium
store depleting agent, thapsigargin (Marshall et al., 2000). The protein
tyrosine kinase (PTK) inhibitor genistein mimicked the response and was
not additive, suggesting that PTK inhibition may be involved. Daidzein is
the inactive control molecule for genistein. Daidzein was ineffective by
itself and did not affect the subsequent response to hypotonic shock.
Taken together, these results imply that hypotonic shock inhibits Cl
William S. Marshall
411
secretion in MR cells by a mechanism involving PTK inhibition. One PTK

that has been shown to be present by immunocytochemistry is focal
adhesion kinase (FAK) (Marshall et al., 2005a); this is of particular
interest because of the association of FAK with integrin-signalling
processes and association of FAK with the cytoskeleton, two possible
linkages with volume sensing mechanisms (Fig. 14.3).
Recently evidence was provided to support involvement of
p38MAPK, n-terminal kinase (JNK), protein kinase C (PKC), oxidative
stress response kinase (OSR1) and ste20 proline rich stress kinase (SPAK).
The drug SB203580 significantly enhanced the decrease in current by
hypotonic shock in killifish opercular membranes suggesting an inhibitory
role of p38 MAPK in the hypotonic inhibition (Marshall et al., 2005a).
Hypotonic stress rapidly and transiently increased phosphorylated p38
MAPK, measured by Western analysis, by eight-fold at 5 minutes, then
more slowly again to seven fold at 60 minutes. Hypertonic shock slowly
increased pp38 by seven fold at 60 minutes. Phosphorylated JNK kinase
was increased by 40-50% by hypotonic shock. The stress protein kinase
SPAK and oxidation stress response kinase OSR1 were present in salt and
fresh water (FW) acclimated gill and opercular epithelium with higher
expression in FW (Marshall et al., 2005a). Using immunocytochemistry,
SPAK, OSR1 and phosphorylated focal adhesion kinase (pFAK-tyrosine,
position 407) colocalizes with NKCC at the basolateral membrane
(Marshall et al., 2005a). Hypotonic shock in a rapid and stable manner
dephosphorylates FAK at position 407 but not at other tyrosines (Y397,
Y576, Y577, Y861), as detected by immunocytochemistry and Western
immunoblots (Marshall et al. 2005b). The protein tyrosine kinase inhibitor
genistein (100 mM) inhibits Cl secretion that was high, increases
Cl secretion that was low and reduces immunocytochemical staining for
phosphorylated FAK. The association of FAK with NKCC regulation is an
interesting new role for this kinase in epithelial transport that deserves
more in-depth investigation (see Fig. 14.3 and Table 14.2).
Cultured gill epithelial cells provide a model for gill cell volume
responses. In cultured rainbow trout pavement cells, hypotonic shock,
applied as two-third strength dilution of Ringer solution produced rapid
cell swelling followed by a slow regulatory volume decrease (RVD)
(Leguen and Prunet, 2004). Hypotonic shock induced a biphasic increase
in intracellular Ca2+ comprising an initial peak followed by a sustained
plateau. Calcium free Ringer had no effect on isotonic cell volume, but
Fig. 14.3 Hypothetical model of basolateral membrane transport in rapid control of Cl secretion. Basolateral regulation of NKCC (Na+,K+,2Cl
cotransport, secretory type, NKCC1) is complex and appears to involve a stable activated state, a stable deactivated state and an unstable
regulating state. NKCC may be activated by cell shrinkage (hypertonic shock, right side) via a cascade involving many kinases (p38, JNK, MLCK
and PKC) linked in some fashion to a final kinase in the chain, SPAK or OSR1 that ultimately phosphorylate NKCC at two serines that, in turn,
activate the transporter. Focal adhesion kinase (FAK) is phosphorylated at tyrosine 407 when NKCC is activated (lower right) and this state is
consistent with regulatory volume increase (RVI). Not shown is the common hormonal pathway for activation via adenylate cyclase, cAMP and
PKA. Hypotonic shock and elevation of intracellular calcium (left side) deactivate the transporter apparently via a cascade involving SRC, ERK1,
p38, JNK and an ultimate protein phosphatase that dephosphorylates NKCC and FAK and turns off the transport, consistent with regulatory
volume decrease (RVD). The order of enzymes in the cascades and the scaffolding role for FAK are hypothetical. The involvement of SRC is
inferred from pathways involving integrin, FAK and SRC in cell volume regulation from mammalian systems. Norepinephrine acts via
a2-adrenergic receptors that are mediated through inositol tris phosphate (IP3) from phosphoinositolbisphosphate (PIP2) and deactivate NKCC
via intracellular calcium. It is not clear what stabilizes the deactivated state of NKCC during regulatory volume decrease.
412
Fish Osmoregulation
William S. Marshall
413
inhibited RVD after hypotonic shock. Ca2+ (Leguen and Prunet, 2004).
Similar results were obtained in cultured cells from freshwater or SW
adapted animals and from freshly isolated gill cells. Thus pavement cells
regulate cell volume, associated with intracellular calcium signalling.
Although low calcium solutions and thapsigargin (that depletes
intracellular stores of calcium) did not diminish the hypotonic response in
opercular membranes (Marshall et al., 2000), it is still likely, considering
the similar origins of MR cells and pavement cells, that cell volume
responses in MR cells is also calcium mediated (see Fig. 14.3).
Hypotonic shock applied to the basolateral side of killifish opercular
epithelia induces also a secondary decrease in transepithelial conductance
that develops over an hour or so. Examination by SEM revealed
significantly fewer exposed apical crypts in hypotonically stressed tissues
and that pavement cells apparently close over MR cells (Daborn et al.,
2001). A similar reversible change in MR cell density occurs in estuarine
mudskippers, Periophthalmus modestus (Sakamoto et al., 2000). It would
seem that regulation of the paracellular leak pathway, localized to leaky
junctions between accessory cells and MR cells (Sardet et al., 1979), is
accomplished by retraction of Cl secreting cells and their covering over
by pavement cells that have tight intercellular junctions (Sardet et al.,
1979; Daborn et al., 2001). Furthermore, the robust actin ring surrounding
the apical crypt of MR cells may be the active motor for the retraction
(Daborn et al., 2001).
Hypertonic Shock
Hypertonic shock has the opposite effect to hypotonic shock and can
increase Cl secretion by 100% or more when NaCl or mannitol 50
100 mOsm/kg is added to the basolateral bathing medium (Zadunaisky
et al., 1995; Hoffmann et al., 2002). In nature, blood plasma osmolality
rises after SW transfer to a peak at 8-16 h and the average increase for
killifish is approximately 40 mOsm/kg (Zadunaisky et al., 1995).
Accompanying a smaller increase in Cl secretion, hypertonic stress of
isolated opercular epithelia from FW acclimated killifish significantly
increases the density of exposed apical crypts (Daborn et al., 2001),
suggesting the emergence of Cl secreting cells. The increase in Cl
secretion in SW adapted opercular membranes could be blocked by
basolaterally added bumetanide and by basolaterally added anion channel
414
Fish Osmoregulation
blockers, DPC (Zadunaisky et al., 1995), NPPB and glibenclamide

(Hoffmann et al., 2002). Hyperosmolarity stimulates the heart and
enhances NaCl extrusion functions at the gill (Ando et al., 2003). In the
teleost intestine, hypertonic shock also activates NKCC family
cotransporters, but in this case to aid NaCl uptake from the posterior
intestine of eels (Ando et al., 2003). Hypertonicity in SW transferred
killifish produces differential gene expression and augmentation of NaCl
secretion in protection of plasma ion levels and expression patterns are
quantitatively different for Northern and Southern populations of killifish
(Scott and Schulte, 2005). Because glibenclamide blocks ATP sensitive
potassium channels in cardiac muscle of teleosts (Paajanen and Vornanen,
2002), it is possible that a secondary action via potassium channels in the
tissue may impede the response to hypertonic shock. Curiously, large doses
of amiloride (2 mM) on the basolateral side blocked the Cl transport
stimulation by hypertonic shock (Zadunaisky et al., 1995), suggesting that
the Na+-H+ exchange and/or intracellular pH may affect the hypertonic
response.
In killifish opercular membranes, hypertonic shock increases
phosphorylation level of p38 MAPK by seven fold after 60 minutes of
hypertonic shock (Marshall et al., 2005a). Phosphorylated JNK kinase was
also increased rapidly by 40-50% after hypertonic shock and remained
elevated after 30 minutes in hypertonic medium. The PKC inhibitor
chelerythrine has no effect on hypotonic inhibition but blocks the
recovery (Hoffmann et al., 2002; Marshall et al., 2005a), indicating PKC
involvement in stimulation and presumably also in NKCC activation by
hypertonicity (Marshall et al., 2005a). Further, myosin light chain kinase
(MLCK) is involved in transport activation in the opercular membrane
(Hoffmann et al., 2002; see Fig. 14.3 and Table 14.2).
The second messenger for the hypertonicity response is unknown
(Table 14.2). The increase in secretion is dependent on calcium in the
bathing solutions (Zadunaisky et al., 1995) but calcium ionophore
ionomycin causes inhibition (Marshall et al., 1993) so calcium instead may
play some permissive role. The increase also is also not dependent on
cAMP and PKA (Zadunaisky et al., 1995; Hoffmann et al., 2002). It is
possible that a diffusible second messenger is not involved and instead the
response is sensed and transporter activation takes place by a direct
stimulation of a kinase cascade by a volume sensing molecular complex
such as that depicted in Figure 14.3.
William S. Marshall
415
There is a rapid elevation of Na+, K+-ATPase activity within three

hours of transfer of killifish to SW that also would augment C secretion
rate (McCormick, 2001). This increase in transport enzyme activity is
dependent on translational and transcriptional processes (McCormick,
2001) but it is not known whether the response is hormone mediated or
dependent on increased plasma osmolality.
Role of Intracellular pH
Measurement of intracellular pH with cytoplasmically trapped fluorophore
BCECF indicated no change in intracellular pH of MR cells even after
profound inhibition of Cl secretion by clonidine (Marshall et al., 1993),
suggesting chloride secretion is not physiologically regulated by
intracellular pH. However, pharmacological manipulation of intracellular
pH has noticeable transport effects. Acidification of the cytosolic
compartment in 5% CO2 and addition of NH4Cl to the basolateral but not
the apical side of the killifish opercular membrane inhibits Cl secretion,
apparently as a secondary effect of acidification of the cytosolic
compartment by ammonium loading (Zadunaisky et al., 1995). The
recovery from this acidification is enhanced by the phorbol ester, phorbol
myristate acetate (PMA), and inhibited partially by basolateral amiloride,
suggesting that PKC activation may stimulate Na+/H+ exchange, raise
intracellular pH and allow Cl secretion to return to normal rates. This
implies intracellular pH may indirectly affect Cl secretion, with acidic
stress being inhibitory. Meanwhile, blockage of the anion exchanger with
DIDS has no effect on resting Cl secretion rate (Marshall et al., 1995) and
did not block the stimulation of Cl secretion by hypertonic shock
(Zadunaisky et al., 1995), suggesting that Cl/HCO3 exchange is not
involved in Cl secretion. However, 0.1 mM DIDS blocks the inhibition of
Cl secretion in response to hypotonic shock (Zadunaisky et al., 1997),
while amiloride was ineffective, again implicating the anion exchanger in
mediation of the osmotic response. More direct evidence is needed to
establish whether intracellular pH is involved in physiological regulation
of Cl transport.
Cytoskeleton and Transporters
Hypotonic and hypertonic shock may be mediated via the cytoskeleton in
stretch activated or inactivated membrane elements. The possible
connection of the CFTR type anion channel and actin cytoskeleton arose
416
Fish Osmoregulation
from an association of cAMP dependent phosphorylation by PKA blocked

by the actin disrupter cytochalasin D (Prat et al., 1995). The F-actin
stabilizing agent phalloidin also inhibits generation of sustained Cl
current in colonic T84 cells (Matthews et al., 1994). Partial disruption of
F-actin activates, while more complete disruption inhibits CFTR function
and renders CFTR refractory to cAMP/PKA activation (Cantiello, 2001).
Now, it is known that CFTR is a nucleus for developing F-actin fibers,
observed by confocal microscopy, and that a direct connection between
the channel protein and actin exists (Chasan et al., 2002). Endocytic
recycling and trafficking of CFTR seems to involve Golgi PDZ binding
proteins (Gentzsch et al., 2003) and the well known PDZ binding site
(Swiatecka-Urban et al., 2002) on the carboxy terminus (-TRL) that is
common to human and killifish (Singer et al., 1998) CFTR proteins.
However, rapid inhibition of CFTR involves syntaxin 1A expressed in
Xenopus oocytes (Peters et al., 1999) or in airway epithelia (Naren et al.,
2000). Current thinking has expanded to include syntaxin 1A-SNARE
complex (Ganeshan et al., 2003) or syntaxin 1A-SNAP-23 complex bound
to the amino terminus of CFTR (Cormet-Boyaka et al., 2002) where
syntaxin 1A binds to both CFTR and the SNAP/SNARE proteins (Chang
et al., 2002; Cormet-Boyaka et al., 2002; Ganeshan et al., 2003). In this
complex, binding of syntaxin 1A directly decreases channel open
probability (Chang et al., 2002). Meanwhile, activation of CFTR can occur
via a complex at the carboxy terminus with the >-adrenoceptor, and ezrin/
radixin/moesin binding phosphoprotein regulated by PKA (Naren et al.,
2003; see Fig 14.2). It is tempting to extrapolate to the physiological
downregulation of Cl secretion in teleost MR cells by syntaxin 1A.
Clearly, a demonstration of syntaxin 1A localization to MR cells and
binding to kfCFTR would be a first step to establishing this relationship.
Important also is the identification by (Kltz et al., 2001) of a 14-3-3 gene
in killifish that encodes for 14-3-3, a protein that is known to regulate ion
channels, transporters and cytoskeleton for cells in changing
environments. Protein 14-3-3 is also upregulated in killifish gills on
transfer of killifish from SW to FW (Kltz et al., 2001), implying the
involvement of this protein in long term FW adaptation. Finally, transfer
of killifish to FW produces physiologically relevant decrease in blood
osmolality of approx. 60 mOsm/kg (Marshall et al., 2000) also activates
three identified MAP-kinases: ERK1, SAPK1 (= Jun N-terminal kinase,
JNK) and SAPK2 (= p38) kinase, as all three show enhanced
phosphorylation by immunoblotting with antibodies specific for the
William S. Marshall
417
phosphorylated form of the enzymes (Kltz and Avila, 2001). Thus

osmosensing in gill cells initiates MAP kinase transduced changes in cell
physiology (see Fig. 14.2; Table 14.2).
Whereas mammalian epithelial cells will not normally experience
changes in basolateral osmolality of the magnitudes tested in cell volume
research, gill epithelial cells of estuarine fish actually are osmotically
stressed when these animals change salinity. Hence, the teleost epithelial
cell responses could be more sensitive and amplified than in mammals,
hence easier to study.
Role of PKC Isozymes
The association of PKC= and PKCA with actin cytoskeleton is
demonstrated by the elegant work of Song et al. (2002) where the PKC
non-specific agonist (PMA) in T84 epithelial cells was shown to activate
first PKCA in actin disruption and stimulation of basolateral endocytosis,
followed by a late effect involving activation of PKC= actin stabilization
and inhibition of endocytosis. The isozymes of PKC that may be involved
in Cl transport regulation in teleosts are unknown. Clearly, the actin-PKC
relationship should be clarified for the teleost system before conclusive
relationships between PKC, F-actin and intracellular Ca2+ can be realized.
A parallel between the teleost system and T84 cells is the inhibitory phase
of Cl transport regulation by acetylcholine in killifish (Mendelsohn et al.,
1981; May and Degnan, 1985) and in T84 cells (Song et al., 2002). The
selective activation of PKCA by carbachol and the stimulation of
endocytosis in the late inhibitory phase (Song et al., 2002) invites
speculation that the inhibitory action of acetylcholine and =-adrenoceptor
agonists may be activation of a PKCA and endocytosis or dephosphorylation of NKCC1. Further, PKC= and PKCA are differentially
translocated to the membrane fraction of cells subjected to hypotonic
shock (Liu et al., 2003), implying that hypotonic shock effects could also
be mediated via PKCA.
NON-GENOMIC EFFECT OF CORTISOL
One possibility that has not been investigated for the gill and opercular
epithelium is that of non-genomic responses to corticosteroids. There are
well-established inhibitory roles for cortisol (distinct from other steroids)
at physiological levels to depress prolactin release by cultured cells from
the rostral pars distalis of tilapia (Borski et al., 2002). These effects are not
418
Fish Osmoregulation
mediated by gene transcription or protein synthesis. The effects develop

rapidly, in 10-20 minutes, and are reversible by removal of the hormone.
Similar to somatostatin actions on prolactin cells, intracellular Ca2+ is
decreased and adenylate cyclase and cAMP are reduced by cortisol (Borski
et al., 2002). Interestingly, the intracellular Ca2+ elevation produced by
hypotonic shock in prolactin cells is reversed by 200 nM cortisol. Given
that cortisol may be elevated in fish during salinity change, the possible
downregulation by decrease in adenylate cyclase or reduction of Ca2+
mediated hypotonic response could either aid or exacerbate (respectively)
ion regulation on entry into FW.
PROPOSED MODEL FOR RAPID REGULATION OF
CHLORIDE TRANSPORT
Some of the mechanisms that enter into the hypothetical model here (Figs.
14.2, 14.3) have been established in mammalian systems and appear with
question marks and some others have been demonstrated to be present in
teleosts. Killifish CFTR, because it shares the carboxy terminus PDZ
binding domain in common with the human, may share some aspects of
PDZ binding and regulation. All vertebrate CFTR proteins share the first
two amino acids at the amino terminus and killifish shares the first six of
ten with the human, suggesting the syntaxin 1A binding is also feasible.
Whereas intracellular Ca2+ is normally associated with activation of Cl
secretion, teleost Cl secreting gill and gill-like systems all have Ca2+mediated inhibition. The point of inversion in the regulatory pathway
could be activation of a phosphatase such as Ca2+ activated protein
phosphatase (PP2B, calcineurin) but the PP2B antagonist FK506 is not
effective in blocking clonidine action in the killifish opercular epithelium
(Marshall, unpublished). Another possibility is the activation of a different
PKC isozyme, such as PKCA that is linked downregulation of membrane
transport.
Rapid regulation of NaCl transport at the gill is necessary in fish that
move often between SW and FW, as is true for many estuarine species.
Estuarine animals entering FW may respond in a graded fashion over a few
hours to this stress. Within minutes of exposure to FW, reflex
catecholamine release from the autonomic system could rapidly inhibit Cl
secretion via =-adrenoceptors, to conserve NaCl. Within 1-3 hours as a
result of passive NaCl loss, plasma hypotonicity (hypotonic shock) would
develop in turn producing sustained inhibition of Cl secretion and
William S. Marshall
419
retraction of Cl cells, thus reducing both active and passive NaCl loss.
When estuarine teleosts return to SW after a few hours, NaCl secretion
may be reinitiated, perhaps stimulated by hypertonicity and by peptide
hormones (AVT, glucagon or urotensin I) and neurotransmitters (VIP and
epinephrine operating through > adrenoceptors). In this way, estuarine
fish may rely mostly on rapid acting means of adjusting NaCl secretion and
passive loss, rather than immediately invoking the more energetically
expensive permanent changes to the gill epithelium.
CONCLUSION
This article examines the mechanism of ion transport by mitochondrionrich MR cells and its rapid regulation in teleost fish. Small estuarine fish
experience rapid shifts in salinity in intertidal microenvironments.
Immediate stress responses mediated by neurotransmitters and the adrenal
medullary homolog rapidly change ion transport rates. There are also
direct effects on MR cells by changes in blood osmolality; hypotonic shock
reduces and hypertonic shock increases NaCl secretion rates. Each
reaction involves cascades of phosphorylation or dephosphorylation that
terminate at the ion transport protein. All these are important immediate
responses that help maintain ion balance in early stages of adaptation
before gene expression produces new proteins or newly differentiated cells.
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+0)26-4
15
Control of Calcium Balance in
Fish
Pedro M. Guerreiro* and Juan Fuentes
INTRODUCTION
Perhaps the most evident function for calcium (Ca2+) in animals is the
formation of a hard body shaping and protective structure, which may
either be external (shell of invertebrates) or internal (skeleton of
vertebrates), and which consists primarily of Ca2+ carbonate and/or Ca2+
phosphate complexes. In teleost fish about 99% of the total body Ca2+ is
incorporated into bone, scales, teeth and otoliths (Flik et al., 1986a;
Wendelaar Bonga and Pang, 1991). However, Ca2+, either ionic or
protein-bound, plays a crucial role in numerous other physiological and
biochemical processes such as muscular contraction, vision, blood
coagulation, regulation of enzymatic reactions, modulation of permeability
Authors address: Molecular and Comparative Endocrinology, Centre of Marine Sciences,
CCMAR, CIMAR Laboratrio Associado, University of Algarve, Campus de Gambelas,
8005-139 Faro, Portugal.
*Corresponding author: E-mail: pmgg@ualg.pt
428
Fish Osmoregulation
and excitability of plasma membranes, neural and intercellular

communication and intracellular signalling (Riccardi, 1999, 2000). It is
also important in the reproductive cycle of many animals, namely fish,
where it is an essential component of vitellogenin, the egg yolk precursor
protein (Yeo and Mugiya, 1997) and large amounts of Ca2+ are also
necessary for the production of the eggshells of many species. The
triggering and maintenance of sperm mobility and function and postfertilization events also require the presence of this ion (Stricker, 1999).
The preservation of such vital functions requires the regulation of
Ca2+, within narrow limits, both in the intracellular media (usually around
100 nM) and in the extracellular fluids (about 1-1.5 mM), since deviations
from the physiological range will rapidly disturb the neural, muscular and
cardiovascular functions, leading eventually to either death or
intermediate stages of tetany (due to lack of Ca2+) or lethargy (caused by
Ca2+ excess) (Mundy, 1990). Fish also have to comply with such limits,
and many of the disturbances caused by Ca2+ disequilibria in tetrapods are
also observable in fish, as is the case of lethargy caused in eels by the
removal of the Corpuscles of Stannius (Flik et al., 1995). Nonetheless,
from the data of several studies (Hanssen et al., 1989; Guerreiro et al.,
2002), it seems clear that fishin relation to mammalsare capable to
tolerate a wider variation between minimum and maximum blood Ca2+
concentration, being able to survive for long periods in hypercalcemic
conditions that would be critical to terrestrial vertebrates (Mundy, 1990),
which may be a reflection of the diversity of habitats they live and have
evolved in.
Teleosts represent approximately half of the extant vertebrate species.
They inhabit an immense variety of freshwater (FW) environments, from
the extremely soft, ion-poor Amazonian rivers (where Ca2+ levels are
usually less than 10 mM; Gonzalez et al., 2002) to the very hard waters that
drain calcareous regions. And obviously, fish are not only found in the
marine environment, where Ca2+ concentration in seawater (SW) is
constant at approximately 10 mM, but also in coastal waters with a wide
variety of salinities that range from mixing brackish waters in river
estuaries to hypersaline situations in evaporating enclosed pools,
comprising a large variation in Ca2+ concentration. Some fish are even
able to thrive in waters of extremely high salinity and Ca2+ levels, and
have been observed swimming in the Dead Sea estuary, in areas where
429
Ca2+ concentration is close to 90 mM (Skadhauge and Lotan, 1974). Not

only have some species adapted to life in these extreme conditions but
several can live in environments where Ca2+ concentration fluctuates
greatly, either seasonally or on a daily basis, and have the capability to
adjust their mechanisms to maintain Ca2+ homeostasis in such potentially
challenging conditions.
Salinity or water softness/hardness are not the only factors relevant in
fish adaptation. Fish species also inhabit either in quite acidic or in rather
alkaline environments, such as the rivers of tropical forest (Gonzalez et al.,
1998, 2002) and several lakes of the African rift region (Wood et al., 1989,
2002; Bergman et al., 2003), respectively, conditions that modulate
speciation and ion availability and change the affinity of transporting
mechanisms and the toxicity of certain ions (Spry and Wiener, 1991).
On land, food and drinking water are the only available source of
Ca2+, making the gastrointestinal tract of tetrapods the major site for Ca2+
uptake. Furthermore, due to the varying amount of Ca2+ in the diet and
the episodic nature of feeding, terrestrial vertebrates have to rely on factors
and mechanisms that by default conserve Ca2+ and have a complex system
of cell types and hormonal factors that allows them to use their internal
skeleton as a Ca2+ reservoir. Fish, howevereven those living in soft
FWare surrounded by a virtually infinite supply of Ca2+, readily
obtainable from the environment. Therefore, in addition to the Ca2+
acquired via the food, aquatic animals have access to the ionic Ca2+
occurring in water, and the mechanisms for Ca2+ uptake and control
reflect these important differences in availability and accessibility.
Either in land or in water, several control mechanisms are at play to
maintain Ca2+ homeostasis, including a range of tissues; organs and cell
types, transporting mechanisms, sensory systems and endocrine factors,
which monitor and influence the regulation of cellular and circulating
Ca2+ concentration. This chapter will comprehensively review the
existent literature to illustrate the particularities of Ca2+ balance in teleost
fish: the tissues and mechanisms involved in uptake and excretion in FW
and marine species and the interaction with ambient Ca2+. The focus will
largely be on the role of endocrine factors that control Ca2+ homeostasis
in fish and the relevant developments that arose in recent years in this
field.
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Fish Osmoregulation
CA2+ EXCHANGE TISSUES AND SENSING

MECHANISMS
Given the close contact of fish with the Ca2+ source, water exposed tissues
with large areas and thin epithelia are adequate and energetically favorable
for Ca2+ exchange. Therefore, in fish the gills, the gut and the integument,
in early larval stages, are of crucial importance for Ca2+ uptake.
Gills
The relevance of the branchial tissue for Ca2+ uptake has been
demonstrated in a large variety of fish species, and the gill epithelia is
probably the most important site for Ca2+ uptake in fish, being those
freshwater, marine or euryhaline species (Fenwick, 1989; Flik et al., 1995,
1996; Perry, 1997; Marshall, 2002; Evans et al., 2005).
The branchial epithelia are in direct contact with the Ca2+ present in
the environment, which in SW is present in concentrations well above
those of the Ca2+ in blood plasma (total Ca2+, including bound and free
fractions, 2-3 mM, ionic Ca2+ around 1-1.5 mM), and in FW in
concentrations similar or lower than these internal levels. In both cases,
fish extract Ca2+ from the water. Evidence from electrochemical studies
(Potts and Hedges, 1991; Witters et al., 1992; Verbost et al., 1993b, 1994;
Marshall et al., 1995) demonstrated that the transepithelial potential in
fish gill is always more positive than the equilibrium potential for Ca2+
across the integument and, therefore, the driving force for passive Ca2+
movement across the gills is directed outwards both in FW and in SW fish
(Flik et al., 1995, 1996). This indicates that the overall branchial Ca2+
uptake does not occur by diffusion via paracellular routes but primarily due
to active transport mediated by a transcellular sequence of events,
including a passive entry step, a cytoplasmatic transport phase and an
active extrusion step (Fig. 15.1).
Branchial localization of Ca2+ uptake. The major sites for Ca2+
transport in gills are the mitochondria-rich (MR) cells, also called chloride
cells or ionocytes (e.g., Zia and McDonald, 1994; Marshall et al., 1995;
Marshall and Bryson, 1998; Marshall, 2002). These specialized epithelial
cells occupy a small fraction of the total gill surface area, largely confined
to the filament epithelium and usually concentrated in the intra-lamellar
regions. In general, only a few MR cells are located in the secondary
lamellae. Such cells are also found abundantly in the opercular membrane,
431
Fig. 15.1 Working model of Ca2+ transport in the FW MR-cell (left) and the SW MR-cell
(right). Solid lines indicate active transport; dashed lines indicate secondary transport or
diffusion across channels or across the cell. Solid circles represent ATPases, grey circles
stand for co-transporters and exchangers, and parallel lines represent ion channels. The
mechanisms for transcellular transport of Ca2+ are similar in both FW and SW. Ca2+ entry
occurs down an electrochemical gradient through an apical voltage-independent Ca2+
channel (ECaC). Within the cell, Ca2+ can be sequestered by cell organelles. Ca2+ en route
binds to Ca2+-binding proteins and is transported across the cytosol. Only residual Ca2+
remains free. Extrusion in the basolateral membrane occurs via a Ca2+-ATPase or a Na+/
Ca2+-exchanger. Secondary transport by the Na+/Ca2+-exchanger depends directly on the
Na+ gradient produced by the Na+/K+-ATPase. In SW, the same gradient can be used by
the Na+/K+/2Cl. (Adapted from Marshall et al., 1998; Marshall, 2002).
a thin epithelia exposed to the water in the fish posterior bucal cavity and
that was also shown to be an important site for Ca2+ uptake (Marshall
et al., 1992, 1995; McCormick et al., 1992; Verbost et al., 1997). In this
sense, the opercular epithelia is often regarded as an extension of the
branchial ion-exchanging tissue, and it has been shown that the
operculum contains 15% of the total branchial Na+/K+-ATPase activity in
tilapia, Oreochromis mossambicus (Wendelaar Bonga et al., 1990), and
accounts for 46% of the total Ca2+ uptake in the killifish, Fundulus
heteroclitus (Marshall et al., 1995). Several other non-branchial areas of the
skin contain MR cells and may be involved in Ca2+ uptake, but the
relative contribution is most probably proportional to the number of these
cells and, therefore, rather small. In larval stages, though, abundant MR
cells can be found in the head and trunk skin, and in fin membranes, and
the contribution for Ca2+ uptake is most probably considerable (see Early
Life Stages).
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Fish Osmoregulation
MR cells have a distinct apical surface, an array of sub-apical vesicles

and extensive tubular systems emanating from the basolateral membrane,
but the most distinctive feature is the abundance of mitochondria,
indicative of an high level of cell activity (Perry, 1997; Marshall and
Bryson, 1998; Marshall, 2002; Evans et al., 2005). The rates of Ca2+
uptake, in vivo and in vitro are well correlated with the numbers and surface
area of MR cells (Marshall et al., 1992; Perry et al., 1992), and
modifications in environmental Ca2+ trigger significant and rapid changes
in the shape, number and distribution of MR cells in several species,
regardless of the initial ion-composition of the water (Mayer-Gostan et al.,
1983; Katoh and Kaneko, 2002; Sakamoto and Ando, 2002; Moron et al.,
2003). Histological data has showed the presence of oxalate-induced Ca2+
precipitates in the surroundings the MR cell apical membrane in goldfish,
Carassius auratus (Ishihara and Mugiya, 1987), and the presence of 45Ca2+
was specifically localized by autoradiography in rainbow trout
(Oncorhynchus mykiss) MR cells (Zia and McDonald, 1994). In addition,
studies in isolated MR cells have demonstrated the importance of the
Na+/Ca2+-exchanger activity, a key player in Ca2+ transport (Li et al.,
1997), and recently, Galvez et al. (2006), have demonstrated differential
Ca2+ transport rates in two functionally distinct populations of rainbow
trout MR-cells and also in gill pavement (PV)-cells in vitro. They showed
that 45Ca2+ uptake was most pronounced in PNA+ (peanut lectin
agglutinin positive) MR cells, with levels over threefold higher than those
found in either PNA- MR or in PV cells, suggesting that the PNA+ MR
cell type is a high-affinity and high-capacity site for apical entry of Ca2+
in the gill epithelium.
Ca2+ uptake in the apical membrane. Intracellular Ca2+ concentration
is quite low (in the submicromolar range (Schoenmakers et al., 1993;
Larsson et al., 1998)) and Ca2+ entry across the apical membrane of the
MR cells is believed to be a passive process, following the concentration
gradient, that occurs even in very soft waters. Due to the detrimental
effects of high intracellular Ca2+ concentrations that could arise from this
gradient difference, especially in SW, it is also expected that this step is
subject to both short- and long-term hormonal regulation. The apical
entry step occurs probably through voltage-insensitive Ca2+ channels, and
their numbers and distribution may make this the rate-limiting step in the
overall Ca2+ uptake process (Flik et al., 1993b; Perry et al., 2003). The
initial studies showed that lanthanum, La3+, a voltage-independent Ca2+channel inhibitor, blocked the entry of Ca2+ in branchial transporting cells
433
(Perry and Flik, 1988) and data gathered by the use of specific L-type Ca2+
channel antagonists verapamil and diltiazem demonstrated that these
were effective in reducing Ca2+ uptake but that nifedipine was without
effect (Bjornsson et al., 1999), making inconclusive the existence or
importance of voltage-sensitive Ca 2+ channels in the uptake process.
More recently Rogers and Wood (2004) re-addressed this issue using both
voltage sensitive and voltage insensitive apical channel blockers. In their
study, Ca2+ uptake by rainbow trout was 55% reduced in the presence of
1 mM La3+ and 74% by a similar concentration of cadmium (Cd2+), while
the voltage-dependent, L-type Ca2+-channel blockers nifedipine and
verapamil did not affect Ca2+ influx. Although more data is necessary to
fully characterize the Ca2+ entry step in the brachial epithelium, this
initial process seems to be chiefly mediated by voltage-independent
channels.
Recently, Qiu and Hogstrand (2004), Pan and colleagues (2005) and
Shahsavarani and co-workers (2006) isolated and cloned a branchial
epithelial Ca2+ channel (ECaC) in the pufferfish Fugu rubripes, the
zebrafish (Danio rerio) and in the rainbow trout, respectively, which has a
sequence similarity to the mammalian non-voltage gated epithelial Ca2+
channels TRPV5 and TRPV6 responsible for Ca2+ reabsorption in the
kidney (Nijenhuis et al., 2005). Functional expression of the Fugu ECaC in
MadinDarby canine kidney (MDCK) cells confirmed that the channel
mediates Ca2+ influx and was also permeable to zinc (Zn2+) (Qiu and
Hogstrand, 2004). Furthermore, the rainbow trout ECaC mRNA and
protein levels respond to treatments known to increase or decrease Ca2+
uptake capacity (Shahsavarani and Perry, 2006). Exposure of fish to soft
water caused a significant increase in ECaC mRNA levels and an increase
in ECaC protein expression, while infusion with CaCl2 was associated with
a significant decrease in ECaC mRNA levels. ECaC mRNA and protein
expression were also increased in fish treated with cortisol, known to
produce hypercalcemic effects. Interestingly, the results of these studies
also demonstrated that the ECaC expression is not confined to the MR
cells but also exists in PV cells that could, thus, potentially contribute to
Ca2+ uptake (see Shahsavarani et al., 2006). Although more functional
characterization and species comparative analysis of the ECaC is needed,
there is evidence that this may be the mechanism responsible for the first
step in branchial Ca2+ uptake.
434
Fish Osmoregulation
Transcelullar Ca2+ transport. Once inside the cell, most Ca2+ is either
sequestered in organelles or diffuses through the cytoplasm probably
bound to Ca2+-binding proteins (CaBPs) (Perry, 1997; Bjornsson et al.,
1999). To date, little focus has been given to this step. Several CaBPs have
been identified in the gills of the channel catfish Heteropneustes fossilis, but
immunolocalization studies indicate that with the exception of
parvalbumin, all seem to be concentrated in neuroendocrine cells (Fasulo
et al., 1998). Fish-specific CaBPs with characteristics similar to calbindin
and S-100, found in Ca2+ transporting tissues of higher vertebrates, were
found in the branchial tissue of the European eel Anguilla anguilla (Hearn
et al., 1978) and the catfish Ictalurus punctatus (Porta et al., 1996), but
there is no functional evidence linking to Ca2+ transport.
Basolateral Ca2+ extrusion. The exit of Ca2+ across the basolateral
membrane is an active, energy-consuming step that occurs against an
electrochemical gradient. This step involves membrane transporters, such
as the Ca2+-ATPase and the Na+/Ca2+-exchanger (Flik and Verbost,
1993; Flik et al., 1996; Perry, 1997; Marshall and Bryson, 1998; Marshall,
2002; Perry et al., 2003). The relative importance of these two-membrane
carriers remains to be fully determined. Earlier studies in eel and tilapia
branchial membrane vesicles isolated a Ca2+-ATPase with high affinity for
Ca2+ (Flik et al., 1983, 1984a, b, 1985a, b) and showed that it was upregulated by hormonal factors that also lead to hypercalcemia (Flik et al.,
1984b, c, 1989; Flik and Perry, 1989). These observations led to the
suggestion that Ca2+-ATPase was responsible for most of the transcellular
Ca2+ uptake (Flik et al., 1985a, b), and comprehensive reviews on the
characterization and kinetics of these mechanisms exist (Flik and Verbost,
1994, 1995; Flik et al., 1995, 1996). However, more recent studies showed
that there is also an important role played by the Na+/Ca2+-exchanger
(Flik et al., 1997). In the isolated opercular membrane of killifish, the
Na+/Ca2+-exchanger accounted for approximately 80-85% of Ca2+
transport (Verbost et al., 1997), which was dramatically reduced when Na+
was removed from the bathing solution, or when ouabain was added. On
the other hand, treatment with vanadate, a Ca2+-ATPase inhibitor had
little effect on Ca2+ uptake rate. Furthermore, the affinity and the number
of these carriers, taken with the Na+/K+-ATPase, would suffice to account
for Ca2+ and sodium homeostasis in FW and SW (Verbost et al., 1994).
However, acclimation of rainbow trout to 70% SW evokes an 8-fold
increase in Ca2+-ATPase and a 5-fold increase in Na+/Ca2+ exchange,
which seem to be far beyond the requirements for transepithelial Ca2+
435
transport (Flik et al., 1997), thus implying the need for still other Ca2+
transport mechanisms. Clearly, more studies are necessary to determine
the importance of these two transporting mechanisms and, in the case of
Na+/Ca2+-exchanger, the full coupled machinery involved in the gradient
production and maintenance. It seems apparent though that different
species, with different adaptation processes, may rely differentially on
either transporter.
Branchial Ca2+ excretion. Although the renal tubule is considered the
primary site for divalent ion excretion, disparate results indicate a possible
branchial role for Ca2+ excretion in fish. Ca2+ loss via the gills happens by
paracellular routes driven by the outward electrochemical force for Ca2+
both in FW and in SW (Perry and Flik, 1988; Verbost et al., 1994; Flik
et al., 1995; Marshall et al., 1995) and is further influenced by the Ca2+
permeability of the intercellular junctions. In the epithelium of the gills of
FW fish, the electrochemical gradient for Ca2+ is generally directed
outwardly and a substantial passive efflux of Ca2+ has been reported (Flik
et al., 1995). In marine fish, despite the transepithelial potential that
prevents passive Ca2+ entry, Ca2+ loss is at first sight unlikely due to the
high concentration in water. However, the ingestion of SW and
subsequent intestinal absorption may present an important Ca2+ load
which can also be excreted by branchial mechanisms. Hickman (1968)
calculated that in the southern flounder (Paralichthys lethostigma), 68% of
the ingested Ca2+ was absorbed, and only 11% of that could be accounted
for by renal loss, and thus the remaining 89% of the Ca2+ taken up would
have to be excreted by the gills. Evans and colleagues (2005) calculated
the Nernst equilibrium electrical potential for Ca2+ in the order of 1015
mV (inside positive), which is probably below that across the marine fish
gill epithelium, leaving a margin for passive branchial excretion of Ca2+.
More information is needed to substantiate this hypothesis.
Disruption of branchial uptake by metals: the example of cadmium, lead and
zinc. Ca2+ uptake can be disrupted by the presence of other metals in water
and diet. This interference can be acute or chronic, moderate or extreme
and lead to imbalance in Ca2+ homeostasis, growth arrest, deformities and
death (Spry and Wiener, 1991; Croke and McDonald, 2002). Deleterious
effects of increasingly common heavy metals found in water basins such as
Cd2+, Zn2+, lead (Pb2+), and others, in Ca2+ uptake are associated with
similarities in ionic size and charge. It is thought that at least some of these
436
Fish Osmoregulation
metals use the apical Ca2+-channel to gain entry in epithelial cells and
establish a competitive interaction with the Ca2+-binding sites in
basolateral membrane transporters (Verbost et al., 1987, 1992;
Schoenmakers and Flik, 1992; Qiu and Hogstrand, 2004; Franklin et al.,
2005; Morgan et al., 2005; Galvez et al., 2006; Niyogi and Wood, 2006).
Increases in water Cd2+ significantly decrease branchial Ca2+ uptake and
increase Cd2+ accumulation (Verbost et al., 1987; Chang et al., 1998;
Niyogi and Wood, 2006). On the other hand, branchial Cd2+ transport
was partially reduced in fish fed with high-Ca2+ diets, which caused
downregulation of the branchial Ca2+ transporting mechanisms used by
Cd2+ (Baldisserotto et al., 2005; Franklin et al., 2005). Since the effect is
based in competitive kinetics, the toxicity of such metals is most severe in
soft waters because of the low availability of Ca2+, as was observed in the
Amazonian teleost, Colossoma macropomum. In this species, exposure to
water-borne Cd2+ induced an average of 42% inhibition in whole body
Ca2+ uptake relative to controls within 3 hours of exposure, which
increased to 91% after 24 hours (Matsuo et al., 2005). Previous
acclimation to elevated Ca2+ concentrations protected fish against acute
Cd2+ influence. There are no known effects of Cd2+ on Ca2+ efflux.
Rogers and Wood (2004) addressed the interactions between waterborne Pb2+ and Ca2+ uptake in FW rainbow trout. Representative doses
of Pb2+ significantly reduce Ca2+ influx, although its effects were not as
pronounced as those of Cd2+. Apical entry in gill cells is greatly inhibited
by addition of La3+ and Cd2+ to the water but not by blockers of voltagegated Ca2+ channels, and Pb2+ accumulation in branchial cells is reduced
by increasing waterborne Ca2+ concentrations. This indicates that Pb2+
enters via the ECaC. In this study, the high-affinity Ca2+-ATPase activity
was not acutely affected but long-term exposure to dissolved Pb2+
significantly reduced the pump activity, indicating a possible noncompetitive component to Pb2+-induced Ca2+ disruption.
Ca2+ uptake is also inhibited when elevated concentrations of Zn2+
are present. As for the other two metals dealt above, the apical entry of
Zn2+ is inhibited by La3+. The putative presence of an active basolateral
transporter for Zn2+ was investigated in vitro on isolated basolateral
membranes from rainbow trout gill cells (Hogstrand et al., 1996). In this
study, there was no evidence of Na+-gradient driven Zn2+ transport but
this metal was found to be an effective blocker of the Ca2+-ATPase. This
inhibition was both of a competitive and a non-competitive nature,
although the competitive component prevailed. Injection of CaCl2,
437
increased water Ca2+ concentrations and elevated dietary Ca2+ all greatly
reduced Ca2+ and Zn2+ uptake (Barron and Albeke, 2000; Hogstrand
et al., 1996; Niyogi and Wood, 2006).
Intestine
In fish, the role of the intestine in Ca2+ uptake is poorly understood, and
differences in Ca2+ uptake at this site occur between FW and SW fish (see
Fig. 15.2). In FW, the amount of Ca2+ that gains entry through drinking
is rather small and the intestinal contribution to total Ca2+ uptake is
frequently considered accessory (Flik et al., 1996), since the estimated
branchial influx suffices for growth and homeostasis (Flik et al., 1995).
Seawater fish drink copiously to compensate for the water loss and
substantial amounts of Ca2+ enter the gut. From this an important part can
be absorbed, and the intestinal contribution for total Ca2+ uptake in
marine fish is relevant.
Studies using different in vivo and in vitro techniques demonstrated a
net absorption of Ca2+ across the marine teleosts intestine, which has
been estimated to be 20% in tilapia (Schoenmakers et al., 1993), 40% in
cod, Gadus morhua (Sundell and Bjornsson, 1988), 70% in the southern
flounder (Hickman, 1968), and 90% and 40% in seabream Sparus auratus
Fig. 15.2 Total and intestinal Ca2+ uptake by tilapia larvae in FW and SW. In SW larvae,
Ca2+ influx is 3.5-fold higher than in FW and the calculated contribution of the intestinal
route amounts to as much as 35% of the total uptake. In FW, the maximum contribution of
the intestine for total Ca2+ uptake is only close to 5%. * indicates significant difference from
the FW group (P<0.05, t-test). See text for details.
438
Fish Osmoregulation
juveniles (Guerreiro et al., 2002) and larvae, respectively. In SW-adapted

tilapia larvae, the intestinal contribution amounts to about 35% of the
total Ca2+ uptake (Fig. 15.2). Overall, these percentages account for up to
30-40% of the total Ca2+ uptake in most studied fish (Flik et al., 1985b,
1990b; Sundell and Bjornsson, 1988; Vonck et al., 1998) and up to 60-70%
of the total Ca2+ uptake in SW adapted seabream juveniles (Guerreiro et
al., 2002). Furthermore, the intestinal contribution may increase in times
of extra need for Ca2+, such as during gonadal maturation (Mugiya and
Watabe, 1977; Sundell and Bjornsson, 1988; Guerreiro et al., 2002).
The cellular routes for Ca2+ uptake in intestine are similar to those in
the gills, and Ca2+ uptake occurring in the intestinal tract is via active
transport mechanisms (Flik et al., 1990b; Schoenmakers et al., 1993).
Nevertheless, considering the leaky nature of the intestinal epithelium and
the particularly high electrochemical gradients, significant paracellular
transport takes place, and this fraction has been estimated as 40% of total
Ca2+ absorption in the intestine in the marine cod (Sundell and
Bjornsson, 1988).
The enterocyte cytoplasm is negatively charged when compared to the
intestinal lumen, and intracellular Ca2+ concentrations are in the
nanomolar range (Schoenmakers et al., 1993; Larsson et al., 1998). The
combination of these two factors allows the Ca2+ to move across the
brush-border membrane and into the enterocyte interior down an
electrochemical gradient, and studies in brush border membrane vesicles
isolated from FW fish indicated that uptake is composed by a saturable and
a non-saturable component (Klaren et al., 1993) and that the entry in
these vesicles could be ATP-dependent, mediated by a P2 purinoceptorcontrolled Ca2+ channel (Klaren et al., 1997). Studies in isolated cod
enterocytes indicated that unlike the MR cell, the enterocyte apical Ca2+
channel is more likely a L-type voltage-dependent Ca2+ channel (Larsson
et al., 1998, 2002). As for MR cells, transport across the cytoplasma may
require CaBPs (Bjornsson et al., 1999). Extrusion from the enterocyte to
the extracellular fluid occurs through the action of basolateral transporters
which include Ca2+-ATPases, but it is mainly Na+-dependent
(Schoenmakers and Flik, 1992; Flik and Verbost, 1993; Schoenmakers
et al., 1993; Larsson et al., 2002). In studies with membrane vesicles, the
simultaneous operation of the Ca2+-ATPase and Na+/Ca2+-exchanger
indicates that the extrusion activity of the exchanger exceeds that of the
439
ATPase (Flik et al., 1990b). In agreement with the importance of the Na+/
Ca2+-exchanger in the intestine, tilapia enterocytes display a much higher
Na+/K+-ATPase activity than tilapia gill cells (Schoenmakers et al., 1993).
Na+/K+-ATPase activity in seabream enterocytes is also considerably
higher than that measured in gills (Diaz et al., 1998; Almansa et al., 2001).
Almansa et al. (2001) also showed heterogeneity in the biochemical
properties of the Na+-K+-ATPase along the intestine of this marine
species. This agrees with the importance and regional distribution of
several of Na+-driven transporters for nutrient uptake, including Na+/
Ca2+-exchanger. Recently Fuentes et al. (2006) using intestinal segments
from distinct regions of the seabream gastrointestinal tract mounted in
Ussing chambers, showed the existence of differences in Ca2+ uptake
along the intestinal tract that reflects the complexity of the calciumtransporting processes of fish intestinal epithelia.
Thus, the importance of intestinal Ca2+ uptake for total Ca2+
accumulation in fish is variable, due to Ca2+ concentration in water, the
presence of chelating mechanisms, and the regional distribution and
activity of Na+/Ca2+-exchanger and Ca2+-ATPase.
The relevance of dietary Ca2+ is variable, depending on the content
of Ca2+ in the food and/or on the environmental Ca2+ levels, and fish
compensate accordingly: when dietary Ca2+ is low or absent, Ca2+
requirements can be compensated by uptake via the gills from the
surrounding medium (Mugiya and Watabe, 1977) and in contrast, if
dietary Ca2+ content becomes high there is a substantial decrease in
branchial Ca2+ uptake (Baldisserotto et al., 2005; Franklin et al., 2005).
Still, the contribution of dietary Ca2+ to fish Ca2+ uptake is uncertain and
several studies suggest fish can live and grow normally on a Ca2+-poor diet
(see Flik et al., 1995). However, in some cichlid and catfish species, growth
was impaired or reduced when the Ca2+ content in the rearing water was
low, and reduced growth has been shown in a variety of species in fullstrength SW when reared on a Ca2+-deficient food (e.g., Hossain and
Furuichi, 2000). Seawater-adapted seabream juveniles fed Ca2+-deficient
for 9 weeks show growth rates comparable to siblings fed on a control diet,
and the same was true for those fed a Ca2+-sufficient diet in low ambient
Ca2+. When both diet and ambient Ca2+ were limiting, seabream
experienced growth arrest, reduced Ca2+ accumulation and had lower
circulating Ca2+ levels (Abbink et al., 2004).
440
Fish Osmoregulation
The intestine of SW fish is an important osmoregulatory organ and

takes up large amounts of (sea) water (c.a. 10 mM Ca2+) to compensate
for the losses that occur via the integument. Most of the water is absorbed
in the anterior gastrointestinal tract, which leads to increased Ca2+
availability in the intestine and concentrations as high as 25 mM have
been measured in SW-adapted tilapia (Klaren et al., 1993) and in the
winter flounder Pleuronectes americanus (Parmelee and Renfro, 1983). The
intestinal epithelium is leaky and such luminal concentrations can greatly
increase diffusional Ca2+ transport. In an apparent measure to prevent
extra Ca2+ loading of the fish, in vitro studies showed that the net Ca2+
transport is reduced in the proximal region in tilapia adapted to SW
compared to those in FW (Schoenmakers et al., 1993). An important
adaptation to reduce Ca2+ activity and availability in the intestine of SW
fish has been proposed: the chelation of ingested Ca2+ by bicarbonate
(HCO3) to produce deposits of calcium carbonate formed by the favouring
high pH and calcium concentration in the intestinal fluid. According to
recent studies (Wilson et al., 2002; Wilson and Grosell, 2003; Taylor and
Grosell, 2006), HCO3 secretion may play a role in preventing excess Ca2+
absorption by precipitating a large fraction of the imbibed Ca2+ as CaCO3.
In European flounder (Platichthys flesus), the increase of intestinal Ca2+ to
20 mM induced a 57% increase of net HCO 3 secretion, and higher Ca2+
concentrations (40 and 70 mM) in ambient SW resulted in reduced
plasma total CO2, probably used to fuel intestinal HCO 3 secretion
(Wilson and Grosell, 2003). As a result, it is proposed that up to 75% of
the Ca2+ in the intestinal fluid is precipitated and then excreted as faeces,
thus reducing disproportionate branchial and/or renal excretion and risk
of renal stone formation. Intestinal Ca2+ excretion in the cod was
estimated to represent 20% of total Ca2+ excretion and 50% of the
extrarenal excretion (Sundell and Bjornsson, 1988), but little is known in
the case of other species.
Kidney
Ca2+ excretion in fish takes place mainly via the urine, and the amount of
renally excreted Ca2+ varies with the Ca2+ concentration in water. In the
American eel (Anguilla rostrata), Schmidt-Nielsen and Renfro (1975)
observed plasma values of 2.410.06 mM Ca2+ in SW (9.8 mM Ca2+)
and 2.630.07 mM in FW (0.7 mM Ca2+), while the concentrations
in urine and the Ca2+ excretion rates were approximately 5 mM and
441
3.8 mmol/kg/h for SW fish and 1mM and 1.8 mmol/kg/h for FW fish,
respectively. Similar results were obtained for the SW and brackish water
adapted winter flounder (Elger et al., 1987) and illustrate the fact that in
the kidney of FW fish, Ca2+ losses are minimized by means of active
tubular reabsorption of ultrafiltered Ca2+ (calculated by Schmidt-Nielsen
and Renfro (1975) to be up to 80% of the amount in plasma in the
American eel), which is demonstrated by the fact that they have a urine/
ultrafiltered plasma ratio of less than 1 (Foster, 1976; Fenwick, 1981).
Despite the lower concentration of Ca2+ in urine due to the highly dilute
urine produced to counteract the osmotic inflow of water, the large volume
produced makes the renal excretion an important fraction (up to 50%
according to Hobe et al. (1984)) of the total Ca2+ excretion. In SW-fish,
the kidney has a Ca2+-excretory function, yet urine flow is reduced to a
minimum. Tubular reabsorption of Ca 2+ is reduced compared to FW
animals and this ion is also secreted (Schmidt-Nielsen and Renfro, 1975;
Renfro, 1978; Renfro et al., 1982; Elger et al., 1987). In fact, the
importance of secretory processes in the SW kidney can be illustrated by
studies in SW and FW-acclimated rainbow trout in which the percentage
of all glomeruli that were perfusing and filtering was only 5% in SW, and
45% in FW (Marshall and Grosell, 2006). Despite the low glomerular
filtration rate and urine production, renal Ca2+ excretion was determined
to account for up to 65% of the total Ca2+ excretion in the marine Atlantic
cod (Bjornsson and Nilsson, 1985).
The mechanisms of renal Ca2+ transport have not received enough
attention and little is known about the pathways used for Ca2+ movement
in the entire fish nephron and across the renal tubule epithelium.
Transepithelial potential (TEP) measurements in the isolated nephron
segments of FW-fish produced variable results, but the epithelium seems
to be impermeable to passive paracellular Ca2+ movements (Cliff and
Beyenbach, 1988), indicating that the processes of reabsorption must
occur transcellularly. In SW fish, negative TEP in the lumen (Beyenbach
et al., 1986; Cliff and Beyenbach, 1988) seems to exclude the possibility for
paracellular excretion. Consequently, renal Ca2+ reabsorption and
secretion must be active processes.
In isolated vesicles from the euryhaline goby Gillichthys mirabilis and
tilapia, Ca2+ transport across the basolateral membrane was found to be an
energy dependent process mediated by the activity of high-affinity Ca2+ATPases (Doneen, 1993; Bijvelds et al., 1995). In G. mirabilis activity of
these pumps was increased when fish were transferred from SW to FW, and
442
Fish Osmoregulation
were higher in FW-acclimated fish and lower in fish exposed to 200% SW

(Doneen, 1993), while tilapia plasma membrane Ca2+ pump activity was
56% lower in preparations from SW-adapted fish than in preparations from
FW-adapted fish (Bijvelds et al., 1995), which is in keeping with the
differences observed in urine Ca2+ concentration, and indicate an
environmental regulation of renal reabsorption. Furthermore, the use of
specific Ca2+-ATPase inhibitors and Ca ionophore A23187 showed that
this pump accounted for 55% of the total Ca2+ transport by the vesicles
and that most of the uptake is ATP-dependent (Bijvelds et al., 1995).
These data establish the importance of basolateral Ca2+-ATPase in the
reabsorption process in fish. On the other hand, and in contrast to the
intestinal mechanism, the involvement of the Na+/Ca2+-exchanger in
renal Ca2+ uptake is unclear. Neither Bijvelds et al. (1995), using vesicles
isolated from the kidneys of FW and SW tilapia, nor Renfro et al. (1982),
in vesicles prepared from the marine winter flounder renal tubules could
find evidence of Na+-dependent Ca2+ transport. It seems then that Ca2+
transport in renal epithelium, either in the reabsorptive or in the secretory
direction, may be fully dependent on ATP-driven pumps.
Studying the secretory process in isolated and sealed renal tubules
from winter flounder, Renfro (1978) showed that they readily accumulated
45
Ca2+ when exposed to increased bath activities of this ion, and that the
kinetics of the Ca2+ uptake process indicate that this accumulation was
not due to the gradient across the tubule wall. In a later study, inhibition
of Ca2+ uptake by La3+ and stimulation by Ca ionophore A23187
suggested that ATP-altered plasma membrane Ca2+ transport, while
experiments using different Na+ concentrations in the incubation media
showed that Ca2+ uptake is not directly related to the bath-to-tissue Na+
gradient (Renfro et al., 1982). Therefore, Ca2+ accumulation in the tubule
lumen (e.g., secretion) is ATP dependent but not Na+ dependent.
Furthermore, the inhibition by La3+ also indicates that Ca2+ entry in the
cell during the secretory process is mediated by a Ca2+-channel.
The mechanism for Ca2+ entry in the ion-transporting kidney cell is
not known. In mammals, Ca2+ enters the renal cell via a highly Ca2+selective channel TRPV5 because of a steep inward electrochemical
gradient across the apical membrane (Nijenhuis et al., 2005). A similar
situation may occur in fish. Due to the high Ca2+ glomerular filtration
rate, a comparable Ca2+ gradient exists, both in FW and SW fish. The fish
kidney express the mRNA for an ECaC (Pan et al., 2005) that has
similarity to the TRPV5-like channel recently found in the branchial cells
443
(Shahsavarani et al., 2006). The mammalian kidney is not capable of Ca2+

secretion and only the apical membrane possesses TRPV5. The fish renal
tubule actively secretes Ca2+, and is likely that a similar channel may also
occur in the basolateral membrane. In fact, studies with aglomerular
species would greatly increase our knowledge on the cellular processes
involved in renal Ca2+ balance.
Due to the extreme importance of Ca2+ as a regulator of intracellular
enzyme activity, all the proposed mechanisms for transepithelial transport
involve the sequestration of Ca2+ en route through the cytosol. A 28 kDa
high-affinity CaBP was found in the kidney cells of the American eel
(Hearn et al., 1978) and an immunoreactivity for an S-100-like protein was
detected in the juxtaglomerular cells of several SW fish but apparently not
in other tracts of the nephron (De Girolamo et al., 2003).
The urine produced by the kidney is retained in the urinary bladder
and its ionic composition is further modified there through transport of
water and/or monovalent ions. A few studies involving the possible
participation of the urinary bladder in Ca2+ balance have been carried out
and suggest that Ca 2+ is not transported across the bladder epithelia
(Foster, 1975, 1976; Renfro, 1975). Reabsorption of water in this tissue
greatly concentrates Ca2+ in the urine, and favours the production of
bladder stones containing Ca2+ salts (Marshall, 1995; Marshall and
Grosell, 2006).
Calcified Structures
Calcified structures make up for the major fraction of Ca2+ in fish (Flik
et al., 1986a; Wendelaar Bonga and Pang, 1991) and life-long growth is
accompanied by long-term net accumulation of Ca2+ into bone and scales.
Although there is still considerable debate on the implications of the
cellular or acellular nature of fish bone (Herrmann-Erlee and Flik, 1989),
and whether it functions as a Ca2+ reservoir to buffer changes in plasma
Ca2+ as it happens in mammals, it has been demonstrated that in periods
of extra demand fish can mobilize Ca2+ from scales and bone (Persson
et al., 1997, 1998; Witten and Villwock, 1997).
Osteoblast- and osteoclast-like cells, the cell types responsible for
Ca2+ deposition and mobilization in calcified tissue are found in both
cellular and acellular bone, and also in scales (Herrmann-Erlee and Flik,
1989), where they are denominated by scleroblasts and scleroclasts,
respectively. However, very little is known about the cellular and
444
Fish Osmoregulation
biochemical processes involved in bone mineralization and regeneration

and the manner in which they relate to Ca2+ balance. Recently, several
studies have addressed these questions using the scales as a model. In
scales, markers for the activity of these cell types, such as the alkaline
phosphatase (ALP), for osteoblast, and tartrate-resistant acid phosphatase
(TRAP) for osteoclast, and the genes involved in extracellular matrix
calcification were shown to vary according to the salinity or Ca2+ contents
of the environment, and/or are under the control of calcitropic endocrine
factors, such as calcitonin, estradiol and parathyroid hormone-related
protein (Persson et al., 1994, 1995, 1997, 1999; Suzuki et al., 2000; Suzuki
and Hattori, 2003; Redruello et al., 2005; Rotllant et al., 2005; Yoshikubo
et al., 2005).
When environmental Ca2+ becomes scarce, mobilization of the
mineral from the skeleton may provide enough Ca2+ for fundamental
physiological processes. Fast-growing juvenile tilapia transferred to lowCa2+ FW showed a decrease in Ca2+ density of bone when compared to
fish kept in normal FW (Flik et al., 1986a). This difference in Ca2+
contents was more evident in skeletal bone (vertebrae and scales; 12%
less) than in dermal bone (operculum, 6-7%). In this study, the authors
calculated the pool of readily exchangeable Ca2+ in the bone of FW fish
to be about 7% of the total hard tissue Ca2+ and about 15% in fish exposed
to low-Ca2+. When Ca2+ uptake in Atlantic salmon was disrupted by
excess of dietary Cd2+, the Ca2+ content of scales was significantly
reduced, and apparently this mobilization was sufficient to maintain Ca2+
circulating levels undisturbed (Berntssen et al., 2003).
Sexual maturation requires enormous amounts of Ca2+ for gonadal
growth and may result in substantial transient resorption from these tissues
(Bjornsson et al., 1999). In fact, mobilization of Ca2+ from scales and the
vertebral skeleton has been demonstrated in salmonids during sexual
maturation and spawning migration from Ca2+-rich SW to FW (Persson
et al., 1998; Kacem et al., 2000). Scale osteoclast activity is increased
during vitellogenesis inducing demineralization of these structures
(Persson et al., 1995). So, when the Ca2+ demand exceeds the capacity of
the Ca2+ uptake mechanisms, or when it is impossible or energetically
unfavourable to extract Ca2+ from the environment, some fish mobilize
Ca2+ from internal stores (Bjornsson et al., 1999). The opposite process,
Ca2+ deposition, has received less attention. Whether fish use the
445
formation of Ca2+ aggregates in bones and scales as a means to reduce

Ca2+ activity in plasma by precipitation is uncertain.
The overall contribution of the mineralized structures for Ca2+
homeostasis is debatable, but it is clear that in some fish they represent an
important source of Ca2+ during critical periods of their life history.
Early Life Stages
Due to the specific characteristics of fish development, a word is due to
Ca2+ uptake during this phase. In early life stages, an adequate supply of
Ca2+ is essential for rapid development of mineralized structures such as
the vertebral column, the fins and the cranium (Faustino and Power, 1998;
1999), which are essential for survival. The relatively few studies on Ca2+
uptake at the larval phase show that this process probably differs slightly
from that of adults (Hwang et al., 1994, 1996; Chang et al., 1997, 1998;
Hwang and Yang, 1997; Guerreiro et al., 2001, 2004b; Chou et al., 2002;
Chen et al., 2003). At these early developmental stages, the classical
organs responsible for Ca2+ transport are not yet completely developed
and in early larval stages alternative and more efficient routes for Ca2+
entry probably exist. Branchial tissue, which in adults accounts for the
great majority of the body surface, is still poorly developed and structures
such as the gill lamellae are only developing secondarily (Sarasquete et al.,
1998). Despite the possibility that gills, with their MR cells, are needed for
ion uptake before they are needed for gas exchange (Rombough, 1999,
2002), their contribution to Ca2+ uptake may not be sufficient for the
requirements of growth; in particular since the vascular system may not be
sufficiently developed to provide developing peripheral bone structures
with Ca2+. It seems likely that in fish larvae MR cells initially found over
the entire body surface (Fig. 15.3) contribute greatly to Ca2+ uptake
(Wales and Tytler, 1996; Wales, 1997; Hiroi et al., 1998, 1999; Van Der
Heijden et al., 1999a; Chang et al., 2001; Kaneko et al., 2002).
The contribution of the developing intestinal tract to Ca2+ uptake in
fish larvae is largely unstudied. However, it has been proposed that it may
be important in early stages (Tytler et al., 1990) by increasing the relative
surface available for ion exchange. The most relevant fraction of Ca2+
uptake in the intestine is most probably related to drinking rather than to
diet, as drinking rates are significantly higher in both FW and SW larvae
compared to juveniles and adults (Perrot et al., 1992; Fuentes and Eddy,
446
Fish Osmoregulation
Fig. 15.3 Confocal laser-scanning micrographs showing fluorescence emitted by the

mitochondria selective probe MitoTracker. It is possible to appreciate distribution and
relative quantity of mitochondria-rich cells in different areas of the body of 30-day-old
seabream larvae: (A) high magnification of the rostral area showing concentration of MRcells; (B) the lower part of the head featuring MR-cells around the eye, jaw and operculum;
(C) detail of the interior epithelia of the opercular membrane showing abundance or MRcells; (D) detail of the posterior area of the head where it is possible to notice high
concentration of fluorescent signal produced by the heart; and (E) detail of the caudal fin
featuring MR-cells in the membranes in between the fin rays.
1997). Although the developing kidney seems to be at least partially

functional from early stages (Nebel et al., 2005), there are no studies on the
contribution of renal transport for Ca2+ balance in fish larvae.
We have estimated the contribution of the extra-intestinal uptake in
seabream larvae to be increasingly important in lower salinities, mostly due
to reduction in drinking and water Ca2+ concentrations (Guerreiro et al.,
2004b). Extra-intestinal uptake was at least 60% of the total Ca2+ entry
measured in full-strength SW more than 85% in 25% SW. Further
reduction in water salinity and Ca2+ content to one-tenth of the original
SW resulted in a decrease in the extra-intestinal influx to 55% of the
original but this route still contributes with at least 94% of the overall
uptake. The contribution from intestinal uptake is difficult to quantify
since absorption rates are unknown. In full-strength seabream larvae, the
drinking rates were reasonably high and a great amount of water enters the
intestine. Roughly 40% to 50% of the 45Ca2+ measured in these larvae at
447
any given time was contained in the intestinal tract. In tilapia larvae, the
contribution of the intestinal fraction was calculated as 35% of the total
uptake, while in FW-adapted fish the proportion resulting from intestinal
absorption was considerably low, averaging only 5% of the overall uptake
(Fig. 15.2).
In previous studies, the effects of environmental Ca2+ concentrations
on the survival, growth, body Ca2+ content and Ca2+ uptake kinetics in
developing tilapia larvae were studied by Hwang and colleagues (1994,
1996) and Chou et al. (2002). They have shown that hatching rates and
growth were similar in larvae from fertilized eggs exposed to either 0.90 or
0.02 mM Ca2+. Body Ca2+ content in low-Ca2+ groups was about 90-95%
that of high-Ca2+ group and exposure to low Ca2+ induced an averaged
1.2-fold stimulation of Ca2+ uptake rates. Ca2+ efflux was also altered, and
after acclimation for 8 days, the effluxes of the low-Ca2+ group were 43%
of the high-Ca2+ group. These studies also show that responses to lowCa2+ environments are age-dependent and when newly hatched-larvae
were transferred to low Ca2+, the net uptake increased (from 5% to 69%)
within 64 hours, while 3-day post-hatched larvae managed to reach the
levels of the control within 38 hours. Declining Ca2+ efflux in 3-day-posthatched larvae occurred 14 hours after exposure, much faster than those
in newly hatched larvae (38 hours). These data indicate that tilapia larvae
are able to modulate their Ca2+ uptake mechanism to maintain normal
body Ca2+ content and rapid growth in environments with different levels
of Ca2+, despite having still quite under-developed Ca2+ exchanging
structures. In fact, these larva are able to increase their Ca2+ content to
about 60 times higher then that of the embryo within ten days after
hatching (Hwang et al., 1994).
Comparison of larva from different fish species also showed differences
in the strategies for Ca2+ balance in response to environmental Ca2+ that
may be associated with different development patterns and environments
in which these fish naturally occur. Seabream larvae, although adapting to
lower Ca2+ environments, show high dependence on environmental Ca2+
for normal uptake (Fig. 15.4) (Guerreiro et al., 2004b), while the studies
conducted on tilapia indicate that these fish easily equilibrate their Ca2+
balance even in low Ca2+ conditions. This was also demonstrated by Chen
et al. (2003) using three FW species, goldfish, zebrafish, and ayu
(Plecoglossus altivelis). While the goldfish larvae were able to maintain
Ca2+ contents in low-, mid-, and high-Ca2+ environments by increasing
448
Fish Osmoregulation
Fig. 15.4 Relationship between whole body calcium uptake and environmental calcium
concentration in seabream larvae transferred from a 100% SW situation. Each point
represents the average and standard error for at least 15 fish exposed to the different Ca2+
concentrations. A highly significant correlation was found. (Adapted from Guerreiro et al.,
2004b).
uptake capability in low-Ca2+ situations, the Ca2+ content of both

zebrafish and ayu larva acclimated to low-Ca2+ were significantly lower
than those acclimated to mid- or high-Ca2+, due to insufficient Ca2+
influx rates.
Sensing MechanismsThe Extracellular Ca2+-Sensing
Receptor (CaSR)
To effectively control Ca2+ balance, vertebrates rely on sensing systems
that monitor extracellular Ca2+ levels. In tetrapods, the modulation of
renal Ca2+ transporting systems and the regulatory calcitropic hormonal
factors are associated with a Ca2+-sensing receptor (CaSR), present in the
cell membrane that binds and is activated by Ca2+ ions in the extracellular
fluids (Tfelt-Hansen and Brown, 2005). This CaSR enables extracellular
Ca2+ concentrations to modulate internal Ca2+ and other secondary
messengers without being taken up by the cell (Riccardi, 2000) and in
tetrapods it is involved in the regulation of parathyroid hormone (PTH)
secretion by the parathyroid gland, responding to minute changes in blood
Ca2+ levels (Tfelt-Hansen and Brown, 2005).
449
Fish also possess such receptors, showing remarkable amino acid

conservation in most domains across species (Flanagan et al., 2002), most
probably due to the specificity of their function. However, the intracellular
domain of the piscine molecule is considerably smaller than that of higher
vertebrates, suggesting that the range of its functions and intracellular
transmission mechanisms may be more limited.
The CaSR mRNA is expressed in several tissues associated to Ca2+
transport, osmoregulation and endocrine secretion. Expression has been
found in the brain, branchial epithelium, kidney, intestine, bone, pituitary,
corpuscles of Stannius of seabream and pufferfish (Ingleton et al., 1999,
2002; Flanagan et al., 2002; Hang et al., 2005; Abbink et al., 2006), and in
addition to these also in the pancreas, muscle and spleen of the rainbow
trout (Radman et al., 2002), and in the caudal neurosecretory system of the
flounder (Ingleton et al., 2002). It was also found in kidney, urinary
bladder, brain, pituitary, gill, stomach and intestine of the tilapia (Loretz
et al., 2004) and in winter flounder and Atlantic salmon (Salmo salar),
there is expression for this receptor in kidney, stomach, intestine, gill, and
brain (Nearing et al., 2002). These expression studies have shown that at
least in intestine and kidney expression levels are affected by
environmental salinity (Nearing et al., 2002; Loretz et al., 2004), indicating
that they may be involved in regulating the response to osmotic and ionic
challenge in fish. Functional work showed that exposure of the lumen of
winter flounder urinary bladder to known CaSR agonists, Gd3+ and
neomycin, reversibly inhibit volume transport, which is important for
euryhaline teleost survival in SW (Nearing et al., 2002) and human cells
transfected with the tilapia CaSR show important intracellular response to
elevations in the extracellular Ca2+ concentration that was dependent on
the ionic strength of the bathing medium, also supporting a role in salinity
sensing (Loretz et al., 2004).
The role of CaSR in the modulation of calciotropic hormones (see
Endocrine Control of Ca2+ Balance) is still quite unknown, but it seems
to be at least involved in the control of stanniocalcin secretion. This factor
serves as an anti-hypercalcemic hormone such that a rise in extracellular
ionic Ca2+ above a physiological set-point evokes an immediate secretory
response (Wagner et al., 1998a). Using pharmacological agents that
increase the sensitivity of the CaSR to Ca2+, Radman et al. (2002) showed
that these had time and dose-dependent stimulatory effects on STC
secretion that were indistinguishable from those of Ca2+ loading. The
450
Fish Osmoregulation
downstream effects of the increased secretion were the inhibition of gill

Ca2+ transport. It is, therefore, predictable that similar modulatory actions
may occur over other calciotropic factors in fish.
The CaSR is also abundantly expressed in the olfactory epithelia of
both FW and SW species (Cao et al., 1998; Flanagan et al., 2002; Hubbard
et al., 2002; Nearing et al., 2002). The presence in this tissue has prompted
the question on its possible function as a proxy for direct determination of
environmental salinity or Ca2+ concentration, that could serve to
forewarn the animal that is reaching the limit of its osmotic tolerance
(Hubbard et al., 2000) and thus select between suitable or less favourable
habitats, or be used to detect landmarks, features of a specific water body,
as earlier suggested by Bodznik (1978) in relation to migrating salmon
returning to their original rivers.
Electrophysiology studies have now shown that the fish olfactory
system is highly sensitive to changes in environmental Ca2+
concentration. Extracellular recordings made from the olfactory nerve of
seabream while the Ca2+ concentration of artificial seawater flowing over
the olfactory epithelium was varied from 10 to 0 mM showed that
reductions in Ca2+ caused a large increase in the firing rate of the olfactory
nerve and further experiments demonstrated that the goldfish olfactory
system is also acutely sensitive to changes in external Ca2+ within the
range that this species is likely to encounter in the wild (0.05-3 mM)
(Hubbard et al., 2000, 2002). In both cases, the response was not due to
the concomitant reduction in osmolality and was specific for Ca2+, and the
apparent EC50 for the response of each species was close to the levels of
their circulating ionic Ca2+ in the plasma.
Given the specificity of the response to Ca2+ and its fitting to
environmental concentrations in the range usually experienced by each
species, a stenohaline non-migratory and a partially euryhaline, it seems
that this olfactory sensing may be a general phenomenon in teleosts and
is likely involved in internal Ca2+ homeostasis. The existence of Ca2+
sensors linked to the MR cells, that could respond to variations in ambient
Ca2+ had already been predicted (Flik and Verbost, 1995), based on the
existence of Ca2+-responsive receptor cells in the skin of fish, and the
proliferation of gill CaBPs in low ambient Ca2+.
Although it seems clear that fish have internal and external CaSRs, it
still needs to be determined if sensing of external Ca2+ levels can trigger
changes in the activity of Ca2+-handling tissues and/or in the secretion of
calciotropic factors.
451
ENDOCRINE CONTROL OF Ca2+ BALANCE

The endocrine system of fish is responsible for maintaining circulating
Ca2+ levels within tight limits, acting to up-regulate or down-regulate the
exchange of Ca2+ between the blood and the environment and between
the blood and the internal stores (see Fig. 15.5). In terrestrial vertebrates,
the parathyroid hormone (PTH) and calcitonin (CT), together with 1,25dihydroxy vitamin D3 (1,25(OH)2D3; the most effective vitamin D3
metabolite) seem to be the main factors controlling extracellular Ca2+
levels, Ca2+ uptake and excretion and bone mobilization (Mundy, 1990;
Mundy and Guise, 1999). PTH is a hypercalcemic factor that acts directly
by stimulating Ca2+ resorption in the bone and reabsorption of Ca2+ in the
kidney. It also stimulates the synthesis of 1,25(OH)2D3 which promotes
intestinal Ca2+ absorption, allowing enough Ca2+ to be taken up by the
organism. 1,25(OH)2D3 is also involved in the up-regulation of plasma
Fig. 15.5 Calcium balance in fish. Illustrative model of the overall interaction between
control factors, transport tissues, sensing mechanisms and external and internal Ca2+
pools. Changes in extracellular Ca2+ are monitored by internal CaSRs that modulate the
secretion and circulating levels hyper- and hypocalcemic factors. These act over the
transporting tissues to promote either and increase in Ca2+ uptake and/or Ca2+ retention or
a reduction in Ca2+ uptake and/or stimulation of Ca2+ excretion. Actions on mineralized
tissues can also be evoked to induce Ca2+ resorption or deposition. Whether the exchange
mechanisms can be triggered directly by changes in environmental Ca2+ via an olfactory
CaSR is uncertain.
452
Fish Osmoregulation
Ca2+ due to its actions in the bone and kidney. CT counteracts the action
of the other two, rapidly preventing Ca2+ release from bone tissue by
promoting osteoblastic and osteocytic activities. It also stimulatesto a
lesser extentCa2+ excretion via the gut and kidney (Mundy, 1990;
Mundy and Guise, 1999).
The establishment of an endocrine model for the control of Ca2+
balance in fish has been a difficult task, mostly due to the fact that the
nature of the actions of many known factors is still inconclusive and to the
constant appearance of possible new players. The range of factors reported
to have direct or indirect influence in Ca2+ metabolism is increasing, and
recently, the arrival of an entire family of heavy weight contenders opens
space for new questions. Hormones with direct effects on Ca2+ balance
include stanniocalcin, calcitonin, the metabolites of the vitamin D3,
estradiol in specific stages of the life cycle, and at least some members of
the recently uncovered piscine PTH/PTHrP family of proteins (Canario
et al., 2006). Other factors promote changes in osmoregulatory, acid-base
and metabolic parameters, and have indirect actions over Ca2+ balance.
Pituitary Hormones and Cortisol
Pituitary hormones are involved in a multitude of functions, and several
reports indicated, with more or less certainty, that the members of the
prolactin (PRL) gene family have calciotropic actions in fish.
Prolactin. The true role of PRL in fish Ca2+ balance is not completely
clear. If on one hand it has direct actions in plasma Ca2+ and Ca2+
transporting mechanisms, on the other the disturbances in Ca2+ balance
per se do not affect its expression and secretion, which seem to be regulated
by osmolality or extracellular Na+ and Cl levels rather than Ca2+ levels
(Wendelaar Bonga and Pang, 1991; Arakawa et al., 1993; Kaneko and
Hirano, 1993; Seale et al., 2003). Moreover, despite early studies showed
that removal of the pituitary leads to hypocalcemia, in most cases the
hypocalcemia is accompanied by decreases in the concentration of other
electrolytes. In these cases, prolactin replacement restores Ca2+ levels but
also those of the remaining electrolytes. In this context, PRL can be
regarded as an osmoregulatory factor that has hypercalcemic actions in
fish.
Ovine PRL administration induces an increase in plasma Ca2+ in
tilapia (Flik et al., 1986b) and carp Cyprinus carpio (Chakraborti and
Mukherjee, 1995), confirming identical observations in other teleosts,
453
including sticklebacks (Gasterosteus aculeatus), American eels, rainbow

trout and the killifish (Flik et al., 1986b). It also induced significant Ca2+
influx and reduction of Ca2+ efflux in tilapia, resulting in net Ca2+
accumulation. The increase in Ca2+ uptake was associated with enhanced
activity of the branchial high-affinity plasma membrane Ca2+-ATPase
(Flik et al., 1984b, 1989). These results were confirmed by the use of
homologous recombinant tilapia prolactins, PRL-I and PRL-II that differ
in size by 11 amino acids (Specker et al., 1985). Both PRLs increased the
activity of the Ca2+-ATPase in a plasma membrane preparation of the
branchial epithelium but dose-response studies demonstrated that PRL-I
was twice as potent as PRL-II in inducing hypercalcemia (Flik et al., 1994).
The ratio between Ca2+-ATPase and Na+/K+-ATPase increases upon
PRL treatment, as does the proliferation of opercular MR cells, but this
increase in cell density may or may not relate specifically to Ca2+
transport. The possible effects of PRL on intestinal Ca2+ balance have not
yet been addressed, although the PRL receptor exists in the fish intestinal
and renal cells (Tse et al., 2000; Sandra et al., 2001). In the intestine as in
gill, prolactin induces proliferation of ionoregulatory cells, upregulation of
specific ion transporters and changes in membrane permeability, which
seems to be the hormones main functions in FW adaptation (Sakamoto
and McCormick, 2006). In isolated preparations of winter flounder kidney
cells, ovine PRL had stimulatory effects on phosphate uptake but the
salmon protein was without effect, and the actions on renal Ca2+ transport
were not tested (Lu et al., 1995).
The action of PRL in Ca2+ balance is slow, and the effects on plasma
take between two to four days to achieve, which contrasts with the
immediate responses to PTH in terrestrial vertebrates. This also supports
the role of this hormone as a FW-adapting factor (McCormick and
Bradshaw, 2006; Sakamoto and McCormick, 2006), rather than a true
hypercalcemic factor. PRL secretion is also regulated by factors such as
estradiol (Brinca et al., 2003) that has reported actions in Ca2+
homeostasis (see ahead: Estradiol 17>), suggesting that it may be a
mediator or an agonist of the actions of this steroid. However, cortisol,
which may have hypercalcemic action, decreases the production of PRL
(Borski et al., 2001), which may be related to their opposite roles in
osmoregulation (McCormick and Bradshaw, 2006; Sakamoto and
McCormick, 2006).
Somatolactin. Somatolactin (SL) is another pituitary hormone with
arguably hypercalcemic effects in fish (Kaneko and Hirano, 1993). The
454
Fish Osmoregulation
structure of this hormone is closely related to both PRL and growth

hormone (GH), and its primary function(s) remain mostly unknown but
seems to be involved in reproduction, acid-base regulation, and stress
responses in fishes (Kaneko, 1996). Initial indication for its putative
calciotropic effects came from the fact that low environmental Ca2+
levelsbut not osmolalityare associated with activation of the SLproducing cells in the pituitary of the rainbow trout leading to increased
mRNA levels as well as plasma SL levels (Kakizawa et al., 1993). Plasma
SL levels were also elevated in association with plasma Ca2+ and
phosphate during acute stress and during exhaustive exercise (Kakizawa
et al., 1995) and the number of SL-immunoreactive cells and the secretory
and synthetic activity increased in parallel with maturation and
vitellogenesis, in an apparent association with increasing Ca2+ plasma
levels (Mousa and Mousa, 2000). These studies only provide indirect
evidence, and the effects of low Ca2+ on SL secretion are slow, not
compatible with a homeostatic factor. In flounder renal cells
administration of SL had no effect on Ca2+ transport, but induced
significant reabsorption of phosphate, an ion closely associated to Ca2+
metabolism (Lu et al., 1995). Clearly, further studies are needed before SL
can be considered a calciotropic factor.
Growth hormone. GH is a factor involved in fish osmoregulation,
favouring the adaptation to SW due to the capacity of this hormone to
increase the number and size of gill MR cells and ion transporters involved
in salt secretion (Sakamoto and McCormick, 2006), which may also reflect
in increased Ca2+ transport. Very few studies have addressed this issue.
Flik et al. (1993a) tested the effects of homologous recombinant tilapia GH
on growth, Ca2+ accumulation and Ca2+ fluxes. GH had no significant
effect on Ca2+ influx from the water, but stimulated Ca2+ accumulation in
parallel to increased growth rates, which was achieved by reduction in
Ca2+ efflux. The Ca2+ plasma levels were unchanged and the analysis of
the Ca2+ contents of mineralized structures, normalized to weight, did not
show difference between control and treated fish. It appears than that the
effects of GH in Ca2+ balance are not direct but the consequence of
accelerated growth rate, and that other factors may have mediated the
reduction in Ca2+ efflux.
In an interesting approach, the involvement of PRL and GH in otolith
and scale calcification was examined using hypophysectomized goldfish
(Shinobu and Mugiya, 1995). As predicted by the previous data,
455
hypophysectomy resulted in hypocalcemia, which was corrected by PRL

replacement therapy, but not by GH, which induced a further reduction in
plasma Ca2+. Ca2+ incorporation into otoliths and scales was also
markedly reduced after hypophysectomy due to low Ca2+ availability.
Interestingly, PRL was ineffective in stopping this reduction but treatment
with GH completely counteracted the reduction in otoliths and scales and
even exceeded the respective sham levels, suggesting that GH may be
relevant for Ca2+ deposition.
Cortisol. This steroid hormone produced in the inter-renal tissue is
related to stress response and also to SW adaptation, being involved in the
control of proliferation and apoptosis of MR cells (Wendelaar Bonga,
1997; McCormick and Bradshaw, 2006). In some situations it has been
associated with hypercalcemic effects, but the true nature and length of
this association are unclear. Cortisol administration was shown to produce
hypercalcemia in coho salmon, Oncorhynchus kisutch (Bjrnsson et al.,
1987) and rainbow trout (Flik and Perry, 1989). Plasma concentrations of
cortisol increased in rainbow trout acclimated to low-Ca2+ FW and
remained elevated for 8 days. Branchial basolateral membrane vesicles
prepared from these fish showed increased Ca2+-transport capacity due to
stimulation of Ca2+-ATPase activity, probably as a result of MR cell
proliferation, according to the authors. Fish in normal FW were treated
with cortisol for 7 days. After this period these fish displayed stimulated
whole body Ca2+ uptake and an increase of the branchial Ca2+ transport
capacity; the combination of these changes resulted in hypercalcemia.
Recently, it has been shown that cortisol is capable of rapidly stimulate gill
Ca2+-ATPase, through non-genomic pathways (Sunny and Oommen,
2001). Treatment with cortisol resulted also in increased mRNA
expression and protein levels of the apical ECaC in rainbow trout
(Shahsavarani and Perry, 2006).
Estradiol 17>
Estradiol 17> (E2) has a marked impact on Ca2+ balance in fish. However,
despite its clear calciotropic effects, E2 can hardly be regarded as true Ca2+
regulating factor, given the intermittency and the specificity of its effects,
the time it takes to act, and the fact that it does not respond to fulfil
housekeeping needs or to re-establish Ca2+ homeostasis. To date, to the
best of our knowledge, there is no evidence that E2 synthesis is regulated
by decreases in either extracellular or environmental Ca2+ concentrations.
456
Fish Osmoregulation
This hormone is the main factor involved in gonadal maturation in

(female) fish and the stimulus for the production of vitellogenin, a Ca2+binding protein essential for egg production. The E2-mediated vitellogenin
induction requires elevated extracellular Ca2+ concentrations (Yeo and
Mugiya, 1997) and significant increases in circulating Ca2+ are associated
with rises in plasma E2 levels (Mugiya and Watabe, 1977; Pang and
Balbontin, 1978; Pandey, 1993; Persson et al., 1997; Srivastav and
Srivastav, 1998; Guerreiro et al., 2002; Guzman et al., 2004).
Although the E2-driven actions occur only seasonally for most species,
they have great impact in Ca2+ balance, which is more pronounced in
species such as the tilapia, with short reproductive cycles. In fact, serum
Ca2+ levels significantly decreased after gonadectomy in females but were
not altered in males, and there was no difference in serum Ca2+ levels
between gonadectomized males and females (Tsai and Wang, 2000).
Treatments with E2 produced hypercalcemia, which was more pronounced
in castrated males than in females, suggesting a differential sensitivity in
estrogen receptors between males and females.
The pathways and targets for the hypercalcemic action of E2 seem to
be two-fold, depending on the species: the rise in circulating E2 leads to an
increase of Ca2+ uptake from the water and Ca2+ mobilization from
mineralized structures such as the scales and skeleton. The relative
importance of these two pathways seems to be species specific, and linked
to the life-strategy and environment they inhabit. In salmonids, E2 induces
Ca2+ resorption from scales and bone (Persson et al., 1994, 1998; Kacem
et al., 1998, 2000; Witten and Hall, 2003), in a process associated with the
spawning migration. Using TRAP as a marker, Persson et al. (1998)
demonstrated that scale osteoclast activity increased throughout sexual
maturation in the Atlantic salmon and that Ca2+ was simultaneously
accumulated in the female gonads, and proposed that the scales are
reabsorbed in order to provide Ca2+ for the growing ovaries. Rainbow
trout injected with E2 (10 mg/kg) showed significantly high plasma Ca2+
values when compared to sham-injected fish. Twenty days after the initial
injection, plasma Ca2+ reached approximately 16 mM. This rise was
accompanied by an increase in scale scleroclast activity and a reduction in
scale Ca2+ contents (Persson et al., 1997). The Ca2+-resorbing actions of
E2 in scales as measured by increased TRAP activity have also been
demonstrated in vitro in other FW and SW species (Suzuki et al., 2000;
Rotllant et al., 2005) and this steroid down-regulates the expression of
457
osteonectin in the goldfish, an extracellular-matrix protein responsible for

Ca2+ mineralization in scales (Lehane et al., 1999).
E2 treatment also stimulates Ca2+ exchange with the water. In rainbow
trout juveniles, Ca2+ influxes were significantly increased by a factor of 1.2
while the effluxes remained unchanged (Persson et al., 1994). In the
marine seabream, injection of 10 mg/kg E2 in coconut-oil implants resulted
in a remarkable increase in plasma Ca2+, a 2.5-fold increase in free Ca2+
and a 10-fold increase in plasma total Ca2+, which reached almost 30mM
15 days after the injection (Guerreiro et al., 2002). In these fish, we have
not observed any effects on scale TRAP activity nor in scale Ca2+
contents, but the whole body Ca2+ uptake increased significantly in
response to E2. Both the intestinal and branchial Ca2+ uptake routes were
stimulated by E2, while there were no observable effects over Ca2+ efflux,
resulting in a significant 31% increase in net Ca2+ uptake.
Despite these evident effects, the way E2 interacts with the
transporting mechanisms or with the bone reabsorbing cells is not clear.
Estrogen receptors are found in scales, bone and intestine (Armour et al.,
1997; Persson et al., 2000; Socorro et al., 2000; Filby and Tyler, 2005; Pinto
et al., 2006), but its presence in gills is uncertain (Persson et al., 2000;
Socorro et al., 2000; Filby and Tyler, 2005; Pinto et al., 2006), and this
suggests that at least part of the action of E2 on Ca2+ homeostasis may be
mediated indirectly via other endocrine factors.
The increasing importance of xenoestrogens in the water, as disrupters
of the normal endocrine function, osmoregulatory capabilities
(McCormick et al., 2005), Ca2+ metabolism (Suzuki and Hattori, 2003;
Suzuki et al., 2003) and reproductive cycle (Christensen et al., 1999; Kirk
et al., 2003) has stimulated several studies on the effects of these
substances that use Ca2+ levels as a reliable indicator of vitellogenesis
(Nagler et al., 1987; Gillespie and de Peyster, 2004).
Calcitonin
Calcitonin (CT) is a protein produced in the ultimobranchial glands of
fish, analogous to the parafollicular C cells in the thyroid gland of
mammals, where it has an established hypocalcemic role (Mundy and
Guise, 1999). The nature of CT role in fish is ambiguous, as a consequence
of the discrepancy of several physiological studies which yielded
inconsistent results upon CT administration, showing hypocalcemia
458
Fish Osmoregulation
(Wendelaar Bonga, 1981; Wales and Barrett, 1983; Fenwick and Lam,
1988; Chakrabarti and Mukherjee, 1993; Mukherjee et al., 2004a),
hypercalcemia (Fouchereau-Peron et al., 1987; Oughterson et al., 1995) or
no effects on blood Ca2+ levels (Yamauchi et al., 1978a; Wendelaar Bonga,
1980; Hirano et al., 1981; Bjornsson and Nilsson, 1985).
Despite the diverse action of CT on circulating Ca2+ levels, there is
additional evidence to support a role consistent with a hypocalcemic
factor. CT had potent inhibitory effect on Ca2+ uptake in the salmon gills
perfused under simulated in vivo condition (Milhaud et al., 1977), and
increased efflux from isolated gills of CT-treated salmon, an effect
accompanied by reduction of plasma Ca2+ (Milhaud et al., 1980). Milet
et al. (1979) reported that Ca2+ influx of ultimobranchialectomized eels
was lower than of controls, with unchanged efflux, and that CT perfusion
induced important decreases in influx and increases in efflux. In addition
to these results, CT injection caused an inhibitory action over whole-body
Ca2+ uptake in young rainbow trout (Wagner et al., 1997b) but whether
it also influenced plasma Ca2+ levels was not reported. Significant
inhibitory effects on Ca2+ uptake were also observed in vivo in the
snakehead Channa punctatus and carp in response to salmon calcitonin,
either in normal tap water or low-Ca2+ water, but not in high-Ca2+ water
(Mukherjee et al., 2004b). In these fish, plasma Ca2+ levels were
considerably reduced by the hormone injection (Mukherjee et al., 2004a).
These results are consistent with the presence of CT receptors in the gill
(Sasayama, 1999; Suzuki et al., 2001). The manner in which these
receptors interact with the Ca2+-transporting mechanisms is unknown.
The presence of CT mRNA and CT immunoreactivity (Martial et al.,
1994; Clark et al., 2002; Hidaka et al., 2004) in branchial cells was also
found, indicating that it may be a local regulator of Ca2+ transport,
activated by yet another factor.
CT secretion has also been shown to be regulated by the extracellular
2+
Ca concentration, although results from different studies are conflicting
(Wendelaar Bonga and Pang, 1991). For instance, infusion of Ca2+ into
the Japanese eel was associated with an increase in plasma CT
immunoreactivity (Sasayama et al., 2002), but transfer from FW to SW,
which also modified plasma Ca2+ concentrations was without effect on CT
levels (Suzuki et al., 1999). Changes of Ca2+ concentrations in the
environment were also unable to trigger any change in CT secretion in cod
(Bjornsson and Deftos, 1985) but the maintenance of immature brown
459
trout (Salmo trutta) in water containing 20 and 50 mM Ca2+ resulted in

a significant reduction of plasma calcitonin and the extent of the decrease
was related to higher levels of environmental Ca2+ (Oughterson et al.,
1995). Srivastav and colleagues (2002) demonstratedusing histological
techniquesthat the CT cells in the ultimobranchial tissue of the mud eel
(Amphipnous cuchia) became hyperactive after a 15-day vitamin D3induced hypercalcemia.
In tetrapods, CT has an important role in protecting the skeleton from
demineralization. In fish, this function is also present. Prolonged
administration of salmon CT to SW eels, provoked a decrease of plasma
Ca2+ and an increase of both the osteoblastic apposition and of the degree
of mineralization of the intercellular matrix in the vertebral bone (Lopez
et al., 1976). Ultimobranchialectomy resulted in a rise in serum Ca2+ levels
and completely stopped the process of vertebral bone osteoblastic
apposition and caused a significant decrease in the degree of
mineralization of the bone organic matrix (Lopez et al., 1976). Similar
actions were observed in the goldfish, where CT increased Ca2+
deposition in the rib bone, pharyngeal bone, and scales of starved but not
of fed animals (Shinozaki and Mugiya, 2000) and CT treatment was also
effective in reducing the plasma activity of enzymes related to bone
turnover and caused a significant increase in skeletal bone Ca2+
concentration in the snakehead (Mukherjee et al., 2004a). In in vitro
bioassays CT suppressed the basal osteoclastic activity in scales of the
nibblerfish (Girella punctata) and goldfish, and when administered in
combination with E2 it reduced or abolished the osteoclastic activity
induced by the steroid (Suzuki et al., 2000).
There is evidence to suggest an interplay between CT and E2 in the
regulation of Ca2+ metabolism. Injections of E2 significantly increase
circulating CT in rainbow trout, Atlantic salmon, and coho salmon
(Bjornsson et al., 1989). Furthermore, plasma CT levels show seasonal
variations, increasing before ovulation in salmonids (Bjornsson et al.,
1986; Norberg et al., 1989) and eels (Yamauchi et al., 1978b), and the
levels of CT in plasma of sockeye salmon, Oncorhynchus nerka (Watts et al.,
1975) and rainbow trout (Fouchereau-Peron et al., 1990) are higher in
females rather than in males during the spawning season. A recent report
demonstrated that E2 has a direct up-regulatory action in CT secretion
acting on estrogen receptors of the ultimobranchial gland in the goldfish
(Suzuki et al., 2004). Given the opposite actions of these two factors on
460
Fish Osmoregulation
bone and scale metabolism, it is possible that the rise in CT may reflect a
preventive role, protecting the skeleton from demineralization.
Vitamin D3
Despite some ambiguous results, there is significant evidence for
physiological actions of vitamin D3 metabolites in Ca2+ balance in fish. A
comprehensive and timely review of the piscine vitamin D3 system was
produced by Sundell and colleagues (1996).
The differential roles of the several related metabolites, vitamin D3,
1,25(OH)2D3, 25(OH)D3 and 24,25(OH)2D3, were tested in FW H.
fossilis in a series of Ca2+ concentrations (Srivastav and Singh, 1992;
Srivastav et al., 1997). Vitamin D3 and 1,25(OH)2D3, injected daily,
consistently and gradually increased serum Ca2+ over a period of 5 days,
which culminated in 30% increase in total plasma regardless of the water
Ca2+ concentration. The other metabolites had little (25(OH)D3) or no
effect (24,25(OH)2D3). Daily injections of 1,25(OH)2D3 also increased
the total plasma Ca2+ (but not ionic Ca2+) by approximately 30% in the
male tilapia at 3 and 5 days after the initial injection (Srivastav et al.,
1998). However, in another study, Rao and Raghuramulu (1999) reported
that none of these metabolites displayed calciotropic activity in this
species.
In the SW cod, daily injections of vitamin D3, 1,25(OH)2D3,
25(OH)D3 and 24,25(OH)2D3 did not produce measurable effects on total
plasma Ca2+, but 1,25(OH)2D3 treatment induced hypercalcemia by
specifically elevating the free ionic Ca2+, the most physiologically relevant
fraction. The effect was detectable within 24 hours and lasted for 5 days
(Sundell et al., 1993). In another marine species, the Antarctic fish
Pagothenia bernacchii, a single injection of vitamin D3 increased both ionic
and total Ca2+ levels in plasma, whereas treatment of 1,25(OH)2D3
evoked a decrease of only the ionized Ca2+, and 25(OH)D3 had no effects
on either fraction of plasma Ca2+ (Fenwick et al., 1994). These results,
showing differences in the modulation of the bound and free Ca2+
fractions in relation to 1,25(OH)2D3 treatment, suggest a possible role of
the steroid in the recruitment of Ca2+-binding proteins.
Similar increases in plasma Ca2+ occurred in American eel injected
daily with either vitamin D3 or 1,25(OH)2D3 for 7 days but only in fed and
not in unfed fish. Intestinal Ca2+ absorption was measured in these
animals using perfused averted gut sacs, and fed fish showed higher Ca2+
461
uptake rates than unfed animals, differences that were further increased by
vitamin D3 or 1,25(OH)2D3 (Fenwick et al., 1984). As for H. fossilis,
treatment with either 24,25(OH)2D3 or vitamin D2 had no effect on
plasma Ca2+ levels nor in intestinal Ca2+ absorption. In a similar study
using goldfish, the hypercalcemic action of daily vitamin D3 injections was
also dependent on the feeding status of the animals, increasing plasma
Ca2+ in animals fed after a long starvation period, but not in those fed daily
or unfed (Fenwick, 1984). Intestinal Ca2+ absorption was approximately
25% higher in vitamin D3-treated fish than in those injected with the
vehicle alone. Identical effects of vitamin D3 and 1,25(OH)2D3 on
intestinal Ca2+ uptake were observed in the FW-adapted European eel and
in tilapia (Chartier et al., 1980; Flik et al., 1982).
In the marine cod, perfusion with 25(OH)D3 stimulated the intestinal
Ca2+ uptake by 65%, whereas 24,25(OH)2D3 reduced it by 36%. In this
bioassay, vitamin D3 and 1,25(OH)2D3 did not affect the Ca2+ flux across
the intestinal mucosa (Sundell and Bjornsson, 1990). In a similar
experiment Larsson et al. (1995) found a rapid (within 10 to 25 min) doserelated down-regulatory effect of 24,25(OH)2D3 in Ca2+ uptake, but no
effects of increasing doses of 1,25(OH)2D3. The outcome of these
experiments lead the authors to suggest that an environmental adaptation
could explain the need for a hypercalcemic metabolite (i.e., 1,25(OH)2D3)
in FW fish and an anti-hypercalcemic (i.e. 24,25(OH)2D3) in SW fish.
The enterocytes of both FW and SW fish have cytoplasmatic or
nuclear receptors for 1,25(OH)2D3 (Bjornsson et al., 1999) but specific
binding to enterocyte basolateral membrane has been demonstrated. The
SW cod basolateral membrane only binds 24,25(OH)2D3, whereas in FW
carp specific binding for both 1,25(OH)2D3 and 24,25(OH)2D3 exists
(Larsson et al., 2001). Furthermore, in FW-adapted rainbow trout,
enterocytes display receptors for 1,25(OH)2D3 in the basolateral
membrane, and respond to specific treatment with increased intracellular
Ca2+ concentrations, but no receptors are found for 24,25(OH)2D3 and no
intracellular response is observed when the cells are incubated with
24,25(OH)2D3. After acclimation to SW, 1,25(OH)2D3 receptors are
down-regulated while specific binding for 24,25(OH)2D3 appears (Larsson
et al., 2003). There is also evidence that 24,25(OH)2D3 prevents the Ca2+
entry in isolated cod enterocytes, decreasing intestinal Ca2+ uptake via
inactivation of L-type Ca2+-channels, whereas 25(OH)D3 but not
1,25(OH)2D3, is responsible for increasing enterocyte Ca2+ transport via
462
Fish Osmoregulation
activation of Na+/Ca2+ exchangers, concurrent with activation of L-type

Ca2+ channels (Larsson et al., 2002). Taken together, these data greatly
support a role of the vitamin D3 system in controlling disturbances in Ca2+
balance by maintaining adequate intestinal Ca2+ absorption.
The actions of this hormonal system in renal Ca2+ balance are
unknown and although vitamin D3 stimulated renal phosphate
reabsorption in eel (see Sundell et al., 1996, for reference), neither
1,25(OH)2D3 nor 1,25(OH)2D3 produced any effect in phosphate
transport across winter flounder tubule cells (Lu et al., 1994).
Finally, the administration of vitamin D3 metabolites to fish also
produces (ambiguous) effects on the metabolism of calcified structures.
Injections of 1,25(OH)2D3 decreased bone mineralization in FW-adapted
eel (Lopez et al., 1977, 1980) and tilapia (Wendelaar Bonga et al., 1983),
but in sexually mature SW-adapted female eels, injections of 1,25(OH)2D3
stimulated osteoblast activity and prevented demineralization, acting in a
similar way to 24,25(OH)2D3, reported to increase osteoblastic activity in
tilapia (Wendelaar Bonga et al., 1983). Since mature female fish display
higher demineralization rates due to the Ca2+ demand during
vitellogenesis, this may be perceived as a protective action of the skeleton,
although more studies are clearly necessary to confirm this possibility.
Overall, it seems clear that the vitamin D3 family has important
functions in Ca2+ metabolism in fish. Two metabolites, vitamin D3 and
1,25(OH)2D3 appear to have a hypercalcemic function mainly by
stimulating intestinal Ca2+ uptake, a process which is blocked or reversed
by 24,25(OH)2D3. Measurable levels of vitamin D3 and 1,25(OH)2D3
have been detected in plasma of FW and SW teleosts and 24,25(OH)2D3
was identified in blood of the marine bluefin tuna (Sundell et al., 1996).
To date, there are no studies indicating whether the regulation of these
metabolites is dependent on extracellular Ca2+ levels but changes in
salinity can alter the cellular response to specific metabolites in the
intestine. Whether purely sensory-mechanisms are responsible for the
transition from the FW- to the SW-vitamin D3 system in the intestine
or there is involvement of other factors is unknown.
Stanniocalcin
Stanniocalcin (STC) is a glycoprotein secreted primarily by the corpuscles
of Stannius (CS), which are associated with renal tissue in teleost and
holostean fishes only (Wendelaar Bonga and Pang, 1991). However, it is
463
Fig. 15.6 Whole body Ca2+ influx rates in response to different concentrations of waterborne parathyroid hormone-related protein (134) PTHrP measured over a 4-h period in
tilapia larvae adapted to freshwater (A) and seawater (B). Each group represents the
means and SE of 1525 fish. * indicates significant difference from the respective control
(P<0.05, one-way ANOVA). Note that scale in freshwater is one-fourth that in seawater.
now evident that several forms of STC exist both in fish and in mammals,
expressed in tissues that are not directly related to the CS, such as the
ovary, kidney, gut and gills, and that it has roles in mineral metabolism,
neural differentiation, reproduction, and even in cancer development
(Wagner and Dimattia, 2006). Genes bearing high homology for STC
variants have also been identified in aquatic invertebrates, which suggest
an ancient lineage for the protein (Gerritsen and Wagner, 2005).
Initial evidence for the possible role of CS in Ca2+ control was
obtained by observations that removal of the corpuscles of Stannius, or
stanniectomy (STX), resulted in an increase of plasma Ca2+ in eels and
464
Fish Osmoregulation
killifish (Fontaine, 1964, 1967; Pang, 1971; Fenwick, 1974), while extracts
of the excised glands were able to restore normocalcemia or induce
hypocalcemia (Pang et al., 1973; Fenwick, 1974; Bailey and Fenwick,
1975; Dubewar and Suryawanshi, 1978). Further studies have shown that
the cell morphology and secretory capability of the CS were determined by
alterations in environmental Ca2+ (Pang and Pang, 1974; Meats et al.,
1978; Aida et al., 1980; Wendelaar Bonga, 1980; reviewed by Wendelaar
Bonga and Pang, 1991).
STC was isolated and characterized from the Atlantic salmon
(Wagner et al., 1986) and the Australian eel, Anguilla australis (Butkus
et al., 1987), and has since been cloned or isolated from several other
salmonids, white sucker (Catostomus commersoni), garpike (Lepisosteus
platyrhynchus), bowfin (Amia calva) and arawana (Osteoglossum
bicirrhosum) among others (see Wagner and Dimattia (2006) for
references). In most fish species, the translated preproSTC is a dimer of
identical polypeptide chains. The prepro-STC monomer in salmonids is
256 aa long and is processed into a mature monomer of 223 residues (Flik
et al., 1990a; Wagner et al., 1998b).
Production of STC mRNA and release of the protein are stimulated
by increased Ca2+ levels either in vitro (Wagner et al., 1989, 1998a; Wagner
and Jaworski, 1994; Ellis and Wagner, 1995) and in vivo (Hanssen et al.,
1991, 1992; Wagner et al., 1991, 1998a). Administration of CaCl2 either
by injection or perfusion leads to an elevation of both ionic and total
plasma Ca2+ resulting in the release of STC from the CS into the blood.
The secretion of stored STC from the incubated CS is also stimulated by
elevated levels of ionic Ca2+ (2.5 mM) but not by concentrations that fall
within or below the normal circulating levels (1.01.5 mM).
Transfer of SW-adapted eels to FW or distilled water showed a
significant 50% drop in plasma STC levels (Hanssen et al., 1992), but in
fully acclimated fish, the STC and extracellular Ca2+ levels were not
different between SW and FW. STC secretion and clearance were 70-75%
higher in SW than in FW, and STC injection-induced hypocalcemia was
more pronounced in SW (Hanssen et al., 1993). A posterior study with
FW and SW salmon has revealed no difference in the sensitivity of STC
cells to Ca2+ and also suggested that the increased demand for STC in
marine fishes is met instead by increased rates of hormone synthesis and
secretion and perhaps by a redistribution of STC receptors (Wagner et al.,
1998a). Taken together, these data indicate that STC metabolism responds
465
to changes in internal and external milieus, to compensate alterations in

Ca2+ levels. The hormone seems to be more active in fish transferred to
SW, probably in order to prevent hypercalcemia.
In the tilapia, enlargement of the CS was observed in sexually mature
females, and the size of the CS increased in parallel with the growth of the
ovaries and the elevation of total and ionic Ca2+ levels. Ovariectomy is
followed by a reduction in the size of the CS and by a reduction in plasma
Ca2+ to levels typical for males, in which gonadectomy does not affect size
or ultrastructure of the CS, or plasma Ca2+ levels (Urasa and Wendelaar
Bonga, 1985). Although there are no indications on the STC levels in
these fish, these observations clearly agree with an anti-hypercalcemic
role.
Secretion of STC is modulated by the CaSR. Radman et al. (2002)
showed that Ca2+-stimulated STC secretion in salmon is mimicked by
calcimimetics that increase the sensitivity of the CaR to Ca2+. The
intraperitoneal administration of NPS R-467, a molecule that acts as a
positive allosteric modulator of the CaR, produced time- and dosedependent stimulatory effects on STC secretion that were virtually
indistinguishable from those of Ca2+ loading experiments. Whether this
secretion led to reductions in circulating Ca2+ was not tested, but the
immediate downstream result was a reduction in branchial Ca2+ uptake.
Previously, cholinergic stimulation of STC had been demonstrated (Cano
et al., 1994; Fenwick et al., 1995), which indicates the existence of two
regulatory pathways for STC secretion: a direct regulation by Ca2+ levels
signalling through CaSRs on the CS STC-secreting cells and a cholinergic
route that may be driven by CaSRs located elsewhere.
The hypocalcemic action of STC is mostly produced by rapid
reductions in Ca2+ influx, and also increases in Ca2+ efflux. Single- and
cumulative-injections induce up to 60% reduction in gill Ca2+ transport
in rainbow trout (Wagner et al., 1997a), and a similar effect was observed
in tilapia. In this fish native STC and a synthetic N-terminal (1-20 amino
acids) fragment were equipotent in reducing Ca2+ uptake (Verbost et al.,
1993a), indicating that this region comprises bioactivity and that these
first amino acids are required for receptor binding and activation.
Increases in Ca2+ uptake after STX were observed in several species,
either in isolated gills or in in vivo experiments (e.g., So and Fenwick, 1977,
1979; Milet et al., 1979; Lafeber et al., 1988; Verbost et al., 1993a, b; Van
Der Heijden et al., 1999b). In eel, removal of the CS induced a 4-fold
466
Fish Osmoregulation
increase in plasma Ca2+, reaching close to 8 mM, the result of a 7-fold

increase in branchial Ca2+ uptake (Van Der Heijden et al., 1999b).
Branchial Ca2+ influx following STX increased during the first four days
and stayed elevated for at least 40 days. Removal of the CS had no effect
over the transepithelial potential across the gill cell, indicating that the
increase of Ca2+ transport occurs across the cells and not in a paracellular
fashion (Verbost et al., 1993b). In this study, the increased Ca2+-influx
after STX was not correlated with changes in ATP-dependent Ca2+extrusion across the basolateral membrane, and STC did not affect the
Ca2+-ATPse in isolated basolateral membranes.
This suggests that in the gill cell STC acts by modulating the
permeability of the apical Ca2+-channel. A signal transduction system
between the two membranes must exist (Wendelaar Bonga and Pang,
1991; Flik and Verbost, 1995) but receptors for STC have not been
described in fish. Assuming that the main target for STC is the apical
Ca2+-channel, inhibition of Ca2+ entry in enterocytes and reduced
reabsorption in renal tubule is to be expected. Given the amount of Ca2+
in SW, and the high drinking rates of marine fish, the intestine of SW fish
is a likely target for a hypocalcemic factor. Stanniectomy increased
drinking rates in SW-adapted eels, thus increasing the amount of Ca2+ in
the gastrointestinal tract and inducing a steeper Ca2+ gradient across the
luminal wall. Conversely, STC decreased Ca2+ uptake in the intestinal
membrane in isolated cod gut preparations in a dose-dependent manner,
reaching a maximum of 23% reduction (Sundell et al., 1992).
Unfortunately, very little information exists on the action of STC in the
intestine and the same holds true for the effects in renal function.
Incubation with the hormone failed to produce any effect on
transepithelial Ca2+ transport across winter flounder proximal tubule cells,
but increased phosphate reabsorption, which could likely depress plasma
Ca2+ levels by promoting deposition (Lu et al., 1994). Measurements of
urine Ca2+ concentration and Ca2+ fractional glomerular filtration rates
prior and upon STC treatment are not available.
A stimulatory action on bone and scale mineralization would also be
in line with the effects of a hypocalcemic factor, but the few report
available on the effects of STC in mammalian bone assays show
contradictory results, either causing Ca2+ resorption in a PTH-like fashion
(Lafeber et al., 1986, 1989) or inhibiting the effects of PTH (Stern et al.,
1991; Yoshiko et al., 1996).
467
PTHrP and PTH-like Peptides

Fish apparently lack an equivalent of the parathyroid gland, and were for
long thought to be devoid of parathyroid hormone (PTH), the major
hypercalcemic factor in terrestrial vertebrates. Nevertheless, several
studies, using heterologous antisera, have historically indicated the
presence of PTH-like immunoreactivity in fish pituitary glands, brain and
plasma (Parsons et al., 1978; Harvey et al., 1987; Kaneko and Pang, 1987;
Pang et al., 1988; Fraser et al., 1991), but only in recent years has the
existence of PTH-like peptides and their receptors in fish been firmly
established, initially with the isolation of PTH/PTH-related protein
receptors in zebrafish (Rubin and Juppner, 1999), the cloning of the
PTHrP cDNA in seabream and the characterization of the gene in
pufferfish (Flanagan et al., 2000; Power et al., 2000). It is now clear that fish
possess PTH and PTHrP like genes and 3 PTH/PTHrP receptors (Rubin
and Juppner, 1999; Canario et al., 2006). PTHrP has recently arisen as a
likely hypercalcemic factor in fish.
PTHrP is a multifunctional factor in mammals (see Clemens et al.,
2001, for a comprehensive review of its functions), first identified in
relation to malignant neoplastic tissues and associated to a disease, the
humoral hypercalcemia of malignancy (Moseley et al., 1987). In fish, the
protein has been characterized in pufferfish, seabream and European
flounder. The piscine mature protein is 125 to 129 amino acids long
according to the species, and the N-terminal 34 amino acids, the known
bioactive region in mammals, share 100% similarity due to conserved
amino acid substitutions. PTHrP and PTH/PTHrP receptors type 1 and 3
(PTH1R and PTH3R) are expressed in many tissues related to Ca2+
transport, such as the branchial, intestinal and renal epithelia, Ca2+
deposition, such as bone, scales and cartilage and also in organs involved
in the endocrine response to ion and osmoregulation, e.g., the pituitary,
the inter-renal and the CS (reviewed in Guerreiro et al., 2007).
In terrestrial vertebrates, PTHrP acts as a paracrine or autocrine
regulator, but its presence in high concentration in fish blood, determined
after the development and validation of an homologous radioimmunoassay
(Rotllant et al., 2003), predicts a classical endocrine function, most
possibly related to Ca2+ metabolism, but to date no producing gland has
been unambiguously identified in fish. A likely candidate, however, is the
pituitary gland, since tissue extracts of seabream pituitary contain
significant levels of the peptide (Rotllant et al., 2003) and PTHrP is also
468
Fish Osmoregulation
released constitutively from European flounder pituitary primary cultures

over a 24-hour period (Worthington et al., 2004b). However, mRNA for
PTHrP and PTH1R were down regulated in the pituitary of seabream
transferred directly from SW to dilute SW or acclimated to low salinities
for a week. The inverse relationship was observed in the gill (Abbink et al.,
2006).
In fully acclimated fish, plasma circulating levels seem to be closely
related to both total and ionic blood Ca2+ (Abbink et al., 2004, 2006). In
juvenile seabream fed on a Ca2+-deficient diet, plasma PTHrP was
significantly increased when compared to fish on a normal diet and fish fed
a normal diet but exposed to lower salinity (from 10.5 mM to 0.7 mM
Ca2+) also showed elevated PTHrP levels in relation to salinity and diet
controls (Abbink et al., 2004). In a series of preliminary studies with the
European flounder, Worthington and colleagues (2003, 2004a) observed
that chronically acclimated SW- and FW-animals had similar plasma levels
of PTHrP and Ca2+, but when kept in deionized water (DIW) for 2 weeks
the levels of PTHrP increased significantly. Additionally, injection of the
Ca2+-chelating agent EGTA lowered plasma Ca2+ and significantly
elevated plasma PTHrP 6-8 hours later. Furthermore, a transfer from SW
to FW induced a rise in PTHrP levels (when compared to fish transferred
from SW to SW) within 24 hours, while the reciprocal change (FW to
SW) slightly decreased the circulating protein at both 4 and 8 hours after
the transfer. Increases in PTHrP immunohistochemistry staining intensity
were observed in flounder kidney, gill and pituitary from animals
acclimated to FW compared to those in SW (Danks et al., 1998). On the
whole, these results suggest that Ca2+ levelsboth in the plasma and in
external sourceseither in the diet or in the environment, are a
significant factor regulating PTHrP secretion in fish, although the
mechanisms involved are not completely clear. The data also indicates
that the hormone may be used for rapid compensations in changing
environments, but also necessary to reduce hypocalcemia in long-term
hypocalcemic conditions. Whether the CaSR, the main modulator of the
PTH response to lowered Ca2+ levels in tetrapods, is involved in the
regulation of PTHrP in fish is not known, but changes in CaSR mRNA
expression parallel those of the PTHrP and the PTH1R (Abbink et al.,
2006).
PTHrP seems to be involved with other factors known to evoke
hypercalcemia. In addition to the rise in Ca2+ influx and plasma Ca2+
469
levels upon E2 administration, we have also observed an increase in

PTHrP levels, that preceded the rise in Ca2+ (Fuentes et al., in press). The
estradiol-induced hypercalcemia was blocked by co-implantation of the
(7-34)PTHrP antagonist, which strongly suggests that the rise in Ca2+
levels is due to PTHrP.
In sturgeon (Acipenser naccarii), single injections of piscine N-terminal
(1-34)PTHrP significantly increased whole Ca2+ uptake while decreasing
Ca2+ efflux and, consequently, plasma Ca2+ levels were up-regulated
within 4 hours and sustained for 24 hours (Fuentes et al., 2007). Exposure
of seabream larva to (1-34)PTHrP in SW also caused a significant and
dose-dependent increase in whole body Ca2+ uptake measured 4 hours
after the addition of the peptide (Guerreiro et al., 2001). This effect was
achieved both by stimulated Ca2+ influx and by a reduction in Ca2+ efflux.
Similar results were obtained with SW-, but not with FW-acclimated
tilapia (Oreochromis mossambicus) larvae (Fig. 15.6A, B), indicating a
differential regulation that may result from variations in endogenous levels
of PTHrP in the two environments. The Ca2+ release rate from SW tilapia
isolated gill cells pre-loaded with 45Ca2+ was accelerated by addition of (134)PTHrP to the culture medium, an effect that occurs within seconds to
a few minutes from the initial exposure, and suggests rapid regulation of
basolateral transporting mechanisms (Fig. 15.7A). In opercular
membranes of seabream mounted in Ussing chambers, the exposure to (134)PTHrP also stimulated 45Ca2+ transport from the apical to the
basolateral side (Fig. 15.7B).
The effects of physiological levels (1-34)PTHrP on intestinal Ca2+
uptake were measured in seabream duodenum, hindgut and rectum
preparations mounted in Ussing chambers under symmetric (saline in both
hemichambers) and asymmetric (saline in basolateral, saline similar to
composition of the intestinal fluid in apical) (Fuentes et al., 2006). When
in symmetric conditions, administration of PTHrP resulted in an increase
Ca2+ uptake in duodenum and hindgut and a reduction in efflux in the
rectum, indicating that different mechanisms are responsive to PTHrP
along the intestine. In control asymmetric conditions, there was a basal
positive net uptake in Ca2+ uptake in all regions. Addition of
(1-34)PTHrP in these conditions increased net Ca2+ uptake 2 to 3 fold in
all regions and decreased slightly the epithelial resistance of the intestinal
epithelium, which may favour an increase of paracellular Ca2+ absorption.
470
Fish Osmoregulation
Fig. 15.7 Effects of PTHrP on calcium transport in branchial tissues. Afast action of
different PTHrP dose on 45Ca2+ release from previously loaded gill cells. Isolated tilapia gill
cells were incubated in culture medium with 45Ca2+. Upon PTHrP treatment Ca2+ extrusion
is enhanced in treated cells in relation to control cells. Baction of PTHrP on mucosal-toserosal Ca2+ transport across the isolated opercular membrane of seabream mounted in
using type chambers. Basal measurements were made in membranes exposed to saline
for 60 minutes. PTHrP was added to the serosal side at this point and the effects evaluated
after 60 minutes more. * indicates significant difference from the control group (P<0.05,
J-test).
Which mechanisms are used by PTHrP to evoke these actions on

branchial and intestinal tissue is unknown. PTH1R was identified in the
gill cells by RT-PCR. In enterocytes, the existence of PTH3R was
determined by RT-PCR and its affinity and transactivational
characteristics determined in radioligand binding and intracellular
signalling studies (Rotllant et al., 2006). The receptor Kd is within the
levels of PTHrP in plasma, indicating that increases in plasma hormone
concentrations can have direct effects in the function of this receptor.
471
PTHrP may be an important regulatory factor in fish kidney. In Ussing

chamber studies (1-34)PTHrP induced a slight increase in Ca2+
reabsorption in winter flounder proximal tubule cells, but the major effect
was observed in phosphate secretion, which was significantly stimulated
(Guerreiro et al., 2004a). Removal of phosphate from the blood reduces
Ca2+ deposition and increases the activity of free Ca2+, as expected from
a hypercalcemic factor.
There is also increasing evidence concerning the possible function of
PTHrP as a factor involved in the resorption of the mineralized tissue.
When treated with (1-34)PTHrP, the seabream scales had reduced mRNA
expression of osteonectin, an important extracellular matrix protein
(Redruello et al., 2005). In addition, 10 nM (1-34)PTHrP, acting via the
PTH1R, was equipotent to 1000 nM E2 in stimulating TRAP activity in
scales (Rotllant et al., 2005). Taken together, these results strongly suggest
a Ca2+ mobilizing action of PTHrP in fish scales.
The data reviewed support the view that PTHrP is a factor with
hypercalcemic actions in fish that may counteract the hypocalcemic
action of STC. The possible calciotropic actions of the novel PTH-like
peptides are still to determine, but given the similarity of the N-terminal,
interaction with the receptors targeted by PTHrP would not be surprising.
At least one of the PTH-like proteins is equipotent to PTHrP in
stimulating Ca2+ uptake in larvae (Canario et al., 2006). So, after a long
search for a hypercalcemic hormone, it is possible that a whole family of
peptides may have such functions.
Perspectives
Given the particular environment that fish inhabit, calcium transport
takes places mainly in three different epithelial tissues the gills, the
intestine and the kidney, although we may also include the skin by the
presence of mitochondria-rich cells. Calcium transport across epithelia
shares a general model that is mostly based on the interaction of epithelial
calcium channels, Ca2+-ATPase and Na+/Ca2+-exchangers. With the aid
of primarily vesicle studies, some of the transporters have been
characterized in several species, and the relative importance in calcium
transport defined. However, with the exception of a few studies, little is
known about the regulation of these mechanisms at the molecular and
endocrine levels. This would be of particular interest in the case of
472
Fish Osmoregulation
stanniocalcin that has been a well-established calcitropic factor for nearly

two decades. However, the particular mechanisms responsible for its
biological action in calcium transport remain elusive.
The fact that calcium availability in the water is high has been a good
reason to propose a model for the endocrine control of calcium balance in
fish based in the action of a single factor with hypocalcemic or antihypercalcemic nature: Stanniocalcin. However, the recent discovery of the
PTH/PTHrP family of peptides in fish and their contribution to calcium
balance with all the characteristics of hypercalcemic factors will probably
mean a reassessment of this model for the endocrine control of calcium
balance in fish. In addition, the interaction and putative cross-regulation
of hypercalcemic and hypocalcemic factors in fish in short term and longterm calcium regulation is an issue that has obviously received little
attention, but ensures new achievements on the understanding of how the
endocrine control of calcium regulation takes place in fish.
Acknowledgements
The authors would like to express their appreciation to Professor A.V.M.
Canario and Professor D.M. Power from CCMar (Faro), and Professor G.
Flik from Radboud University (Nijmegen), for their constant support and
encouragement. Unpublished results presented in this chapter have been
funded by projects from the Commission of the European Union, Quality
of Life and Management of Living Resources specific RTD programme
(Q5RS-2001-02904) and Fundao para a Cincia e a Tecnologia (FCT),
Ministry of Science, Portugal (POCTI/ CVT/48946/2002). P. G. is funded
by the Ministry of Science, Portugal through grant SFRH/BPD/9464/02.
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Fish Osmoregulation
+0)26-4
$
Role of Prolactin, Growth
Hormone, Insulin-like Growth
Factor I and Cortisol in Teleost
Osmoregulation
Juan Miguel Mancera1 and Stephen D. McCormick2
INTRODUCTION
Maintenance of constant cellular ion concentrations is a basic
requirement of all life forms. The strategy evolved by teleost fish to achieve
this requirement is by maintaining nearly constant levels of extracellular
ions at approximately one-third the ionic strength of seawater (SW). In
freshwater (FW), teleosts must counteract the passive loss of ions and gain
of water by actively taking up ions (primarily through the gills), and
removing excess water by excreting a dilute urine. In SW, teleosts
Authors addresses: 1Departamento de Biologa, Facultad de Ciencias del Mar y Ambientales,
Universidad de Cdiz, 11510 Puerto Real, Cdiz, Spain.
E-mail: juanmiguel.mancera@uca.es
2
USGS, Conte Anadromous Fish Research Center, Turners Falls, MA, USA.
E-mail: steve_mccormick@usgs.gov
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Fish Osmoregulation
counteract the gain of ions and loss of water by drinking SW, absorbing
water and ions through the gut, and secreting excess monovalent ions
through the gills and divalent ions through the kidney. Details of these
mechanisms can be found in excellent reviews published in the last several
years (Marshall, 2002; Evans et al., 2005).
The demands on these ion-regulatory pathways will change as a
function of environmental salinity, feeding, activity, injury, reproductive
state and a variety of stressors. Therefore, control of ion regulation is
critical, and the neuroendocrine system is the major means for regulating
these mechanisms. Several excellent reviews on various aspects of the
hormonal control of osmoregulation in fish have been published previously
(Foskett et al., 1983; Mayer-Gostan et al., 1987; Bern and Madsen, 1992;
McCormick, 1995, 2001; Sakamoto et al., 2001; Sakamoto and
McCormick, 2006). Here, we will focus on the endocrine mechanisms that
control the overall capacity of the ion regulatory mechanisms in teleost
fish, focussing on the osmoregulatory actions of prolactin (PRL), the
growth hormone (GH)/insulin-like growth factor I (IGF-I) axis and
cortisol. We will build on existing reviews and incorporate new data to give
an integrative synthesis of the role of these hormones in the
osmoregulation of teleost fish.
PROLACTIN (PRL)
PRL is a pleiotropic hormone with a wide spectrum of functions in
vertebrates. Many of these functions are related to osmoregulatory
processes (Bole-Feysot et al., 1998; Sakamoto et al., 2003; Harris et al.,
2004). The first evidence of the hyperosmoregulatory role of PRL in fish
came from the studies by Grace Pickford and her collaborators (1959,
1970). Using hypophysectomized FW-adapted killifish, Fundulus
heteroclitus, they demonstrated that PRL treatment was essential for
survival of this species in a hypoosmotic environment. Although pituitary
PRL is not necessary for FW survival of all teleosts, subsequent studies
have established the hyperosmoregulatory role of PRL using other species,
types of studies, and experimental approaches (see Hirano, 1986;
McCormick, 1995; Manzon, 2002).
PRL has been shown to regulate several aspects of the ion regulatory
mechanisms that are characteristic of FW fish. Water permeability of the
gill, gut, and kidney are generally lower in FW- than in SW-acclimated
fish, and PRL decreased water permeability in these tissues in several
499
teleost species (Table 16.1; see also Manzon, 2002). To date, the
mechanisms and gene products responsible for the actions of PRL on water
permeability have not been identified, though they are likely to include
regulation of tight junctions, membrane composition, and water channels
such as aquaporins.
Treatment with PRL increases the ion uptake capacity of teleosts, and
it is likely that this effect is carried out through regulation of gill chloride
Table 16.1
Physiological evidence for a hyperosmoregulatory role of PRL in teleosts.
Action
Pituitary
Higher PRL cells activity, synthesis and secretion in FW and BW
relative to SW
References
Low osmolality stimulates pituitary PRL secretion in vitro
Nishioka et al. (1988)

Mancera et al. (1993)
Martin et al. (1999)
Seale et al. (2003)
Plasma
Higher PRL plasma levels in FW and BW relative to SW
Manzon (2002)
Receptors
PRL receptor mRNA levels show a negative relationship with
salinity (i.e., lower in higher salinities)
PRL receptors present in gill chloride cells and in kidney
Gills
Exogenous PRL reduces gill Na+,K+-ATPase activity and mRNA
levels
Exogenous PRL stimulates development of chloride cells
fresh water morphology
Kidney
Exogenous PRL increases Na+ reabsorption and water excretion,
through stimulation of glomerular size and urine output
Contradictory effects on renal Na +,K+-ATPase activity, with
increases or no effects
Shiraishi et al. (1999)

Sandra et al. (2000)
Ng et al. (1991)
Weng et al. (1997)
Santos et al. (2001)
Sakamoto et al. (1997)
Kelly et al. (1999)
Herndon et al. (1991)
Pisam et al. (1993)
Clarke and Bern (1980)
Braun and Dantlzler (1987)
Pickford et al. (1970)
Seidelin and Madsen (1997)
Kelly et al. (1999)
Intestine
Exogenous PRL decreases permeability to water and ions and
Na +,K +-ATPase activity
Contradictory effects on intestinal Na+,K+-ATPase activity,
with increases or no effects
Collie and Hirano (1987)

Manzon (2002)
Kelly et al. (1999)
Skin
Exogenous PRL increases mucus production by stimulation of
differentiation and proliferation of mucous cells
Clarke and Bern (1980)

Brown and Brown (1987)
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Fish Osmoregulation
cells. Herndon et al. (1991) found that PRL injection in SW-acclimated

tilapia resulted in decreased chloride cell size, typical of FW-acclimated
tilapia. In the Nile tilapia, Pisam et al. (1993) found that treatment with
PRL increased the number of b-cells typical of FW-acclimated tilapia and
decreased the number of a-cells typical of SW-acclimated tilapia. Kelly
et al. (1999) have found that the impact of PRL on chloride cells of Sparus
sarba is dependent on the environmental salinity; in hypoosmotic brackish
water PRL reduces chloride cell number and size, whereas in SW this
hormone has no effect. Sakamoto and McCormick (2006) have suggested
that the control of cell turnover (apoptosis and cell proliferation) in
different osmoregulatory epithelia (e.g., gill and gastrointestinal tract) is a
critical feature of the control of osmoregulation by PRL.
It is also likely that PRL affects the transporters that are involved in
ion uptake. However, there is still some uncertainty regarding the
transporters that are most directly involved in ion uptake in teleost fish.
To date, the most favored models include a chloride-bicarbonate
exchanger through which chloride uptake is driven through production of
carbon dioxide. Sodium is thought to be taken up through an apical
sodium channel energized by an apical H+-ATPase. Characterization and
localization of these necessary transporters to fully validate these models
is ongoing, and no information on the role of PRL in regulating these
transporters is currently available. This hormone has variable effects on
gill Na+,K+-ATPase activity among teleost species (see McCormick,
1995). This may stem, in part, from differences in the relative importance
of the Na+,K+-ATPase pump in ion uptake among teleosts (in most
teleosts gill Na+,K+-ATPase activity is higher in SW, but in others it is
lower), their relative euryhalinity, and the salinity at which the studies
were carried out.
The activity of PRL cells is under hypothalamic and extrahypothalamic control. Decreases in plasma osmolality result in increased
PRL synthesis and release (Seale et al., 2003). In addition, other hormones
such as cortisol decrease PRL release (Borski et al., 2002). At the
hypothalamic level, dopamine has a clear inhibitory effect on PRL cells
(Nishioka et al., 1988). In mammals, a specific prolactin-releasing
hormone peptide (Pr-RP) has been described, and in recent years, a Pr-RP
has also been identified in teleosts (see Sakamoto et al., 2003, 2005;
Fujimoto et al., 2006). This peptide is synthesized in hypothalamic
501
neurons, with axons ending close to PRL cells in the rostral pars distalis of
the pituitary (Sakamoto et al., 2003). This peptide can stimulate PRL cells,
increasing synthesis and release of this hormone to systemic blood. In
addition, in the amphibious, euryhaline mudskipper (Periophthalmus
modestus) molecular studies have demonstrated a strong relationship
between expression of Pr-RP and environmental salinity, with higher PrRP expression in fish acclimated to FW and terrestrial environments
relative to SW conditions (Sakamoto et al., 2005). The presence of Pr-RP
in peripheral organs (like gut mucus cells) suggests the possibility of other
actions, including effects on hormone expression outside of the pituitary,
and even direct actions on osmoregulatory tissues (see Sakamoto and
McCormick, 2006).
GROWTH HORMONE (GH)/INSULIN-LIKE GROWTH
FACTOR I (IGF-I) AXIS
GH is a member of the GH/PRL family with a role in osmotic acclimation
(McCormick, 1995) as well as growth and energy metabolism in fish
(Bjrnsson, 1997). GH causes both local and systemic production of IGFI, the latter being produced primarily in the liver. IGF-I carries out many
of the growth-promoting actions of GH, though GH can also have direct
actions on target tissues. Also, in carrying out its osmoregulatory function
in fish, GH appears to workat least in partby increasing circulating
IGF-I and production of IGF-I by the target tissue itself (Sakamoto and
Hirano, 1993).
Smith (1956) was the first to demonstrate that GH treatment
increased the capacity of trout to move from FW to SW. Later, Bolton et al.
(1987) showed that these effects were relatively rapid and independent of
the growth promoting actions of GH. McCormick et al. (1991)
demonstrated that IGF-I was as potent as GH in increasing the salinity
tolerance of rainbow trout. Increased salinity tolerance in response to GH
treatment has also been observed in several non-salmonid teleosts,
including tilapia and killifish (Mancera and McCormick, 1998a, 1999).
GH and IGF-I impacts on hypoosmoregulatory tissue are exerted in
part through their influence on gill chloride cells. Many studies of
salmonids have shown an effect of GH and/or IGF-I treatment on the
number, size and specific ultrastructural features of gill chloride cells (see
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Fish Osmoregulation
references in McCormick, 2001). Sakamoto and McCormick (2006) have

hypothesized that this impact of GH and IGF-I may be through the control
of cell turnover and differentiation in the gill. This effect would be
consistent with the known proliferative and anti-apoptotic roles of IGF-I
in many vertebrate tissues (Wood et al., 2005). It should be noted,
however, that such effects have yet to be demonstrated in osmoregulatory
tissues of fish.
GH and IGF-I are also involved in the upregulation of transporters
critical to salt secretion by the gill. Both Na+,K+-ATPase and the
Na+,K+,2Cl cotransporter (NKCC) are upregulated by GH (Pelis and
McCormick, 2001). Although GH has not been shown to have direct (in
vitro) effects on these transporters, IGF-I increased gill Na+,K+-ATPase,
both in vivo and in vitro (Madsen and Bern, 1993; Seidelin and Madsen,
1999). These impacts on specific transporters may be part of a proliferation
and differentiation pathway for the development of salt secreting chloride
cells in the gill. Surprisingly, the impact of GH and IGF-I on
osmoregulatory tissues other than the gill has received little attention. To
date, what is known suggests that the action of GH and IGF-I on salt
secretory capacity is primarily through its impact on gill physiology
(Seidelin and Madsen, 1999).
IGF-I binding proteins are known to play a critical role in regulating
the interaction of IGF-I with its receptor (Wood et al., 2005). Recently,
Shepherd et al. (2005) have shown that plasma levels of three IGF binding
proteins (21-, 42- and 50-kDa) are higher after salinity acclimation. To our
knowledge, this is the only report of the possible role of binding proteins
in ion regulation. High-affinity, low-capacity IGF-I binding sites
characteristic of receptors have recently been found in Atlantic salmon
gill, and are most abundant in gill chloride cells (McCormick, unpublished
results). Reinecke et al. (1997) present evidence that local production of
IGF-I in the gill occurs primarily in chloride cells (Tables 16.2-16.4).
CORTISOL
Cortisol is the major corticosteroid produced by the interrenal tissue of
teleost fish. This hormone has several established physiological roles
related to osmoregulation, intermediary metabolism, growth, stress and
immune function (Wendelaar Bonga, 1997; Mommsen et al., 1999).
Evidence for the osmoregulatory role of cortisol in fish has been compiled
in excellent reviews (McCormick, 1995, 2001; Sakamoto et al., 2001;
503
Table 16.2 Physiological evidence for a hyperosmoregulatory role of GH in salmonids.

Action
References
Pituitary
Higher GH cell activity, synthesis and secretion in SW
relative to FW

Bjrnsson (1997)
Plasma
Higher plasma GH levels and metabolic clearance rate of GH
during smolting and after transfer from FW to SW

Bjrnsson (1997)
Receptors
GH receptors present at high levels in gill, kidney and intestine
Sakamoto and Hirano (1991)
Gills
Exogenous GH increases gill Na +,K +-ATPase activity and
mRNA levels
Exogenous GH Stimulates proliferation of chloride cells with

seawater morphology
Exogenous GH increases abundance of Na+-K+-2Cl
cotransporter
Kidney
GH treatment has not effect on kidney Na+-K+-ATPase activity
Intestine
Exogenous GH induces seawater morphology in the mucosa of
the middle intestine of Salmo salar previous to smoltification
Exogenous GH increases the drinking response in S. salar
pre-smolts after transfer to SW
Boeuf et al. (1994)

Madsen et al. (1995)
McCormick (1995)
See McCormick (1995)
Sakamoto and McCormick
(2006)
Pelis and McCormick (2001)
Madsen et al. (1995)

Nonnotte et al. (1995)
Fuentes and Eddy (1997)
Evans, 2002). However, in recent years, new aspects of the physiology of

cortisol in fish have arisen, and it is on these that we will focus our
attention.
This hormone is considered a classical SW-promoting hormone, and
evidence has shown a hypoosmoregulatory role of cortisol in several
teleosts. Cortisol decreased plasma ion levels and osmolality in SWadapted teleosts and enhanced salinity tolerance after transfer from lowsalinity water to high-salinity water. This effect is due to increases in gill
chloride cell size and density induced by cortisol treatment (McCormick,
1995, 2001). In addition, this hormone enhanced expression of gill
Na+,K+-ATPase a-subunit and gill Na+,K+-ATPase activity in salmonid
and no-salmonid species (Madsen et al., 1995; Seidelin et al., 1999;
504
Fish Osmoregulation
Table 16.3 Physiological evidence for an osmoregulatory role of GH in non-salmonids.

Action
Pituitary
GH cells activation is depending on the species studied and
the environmental salinity
Plasma
GH levels behave differently depending on the species studied
and the environmental salinity
Receptors
GH binding found in renal tubule of gilthead sea bream
Gill and operculum
Exogenous GH increases salinity tolerance, opercular chloride
cell number and gill Na+,K+-ATPase activity in tilapia
(O. mossambicus) and mummichog (Fundulus heteroclitus)
Exogenous GH does not cause any significant changes in gill
Na+,K +-ATPase activity or a- and b-subunit mRNA levels
in silver sea bream (Sparus sarba)
Exogenous GH increases gill Na+,K +-ATPase activity in
gilthead seabream (Sparus aurata)
Kidney
Exogenous GH reduces Na+,K+-ATPase activity in SW- and
BW-acclimated silver seabream (Sparus sarba)
References
Mancera and McCormick
(1998b)
(1998b)
Munoz-Cueto et al. (1996)
Flik et al. (1993)
Xu et al. (1998)
(1998a)
Deane et al. (1999)
Kelly et al. (1999)
(2006)
Kelly et al. (1999)
Mancera et al., 2002; Laiz-Carrin et al., 2003). Finally, cortisol stimulated

expression and abundance of Na+-K+-2Cl cotransporter in the gills of
FW-acclimated S. salar (Pelis and McCormick, 2001).
At the intestinal level, cortisol stimulated Na+,K+-ATPase activity,
together with ion and water absorption, thus helping adaptation to high
environmental salinity (Veillette and Young, 2005). Also, an improved
drinking response after transfer to SW has been observed in Oncorhynchus
mykiss and S. salar treated with this hormone (Fuentes et al., 1996).
In addition to the classical hypoosmoregulatory role of cortisol, and
according to several evidences (see Table 16.5), a new role of this hormone
either in ion uptake in FW- or BW-adapted fish has been suggested.
McCormick (2001), in his excellent revision of this topic, proposed a dual
osmoregulatory role for cortisol: (1) a stimulatory action on ion secretion
in cooperation with GH/IGF-I axis in hyperosmotic environments; and
(2) an increase of ion uptake in cooperation with PRL in hypoosmotic
environments.
505
Table 16.4 Physiological evidence for an osmoregulatory role of IGF-I in salmonids and
non-salmonids.
Action
References
Plasma
IGF-I levels increased during smolting and SW acclimation
IGF-I binding proteins levels are altered after SW exposure of
rainbow trout

Shepherd et al. (2005)
Receptors
High affinity, low capacity IGF-I binding in salmon gill
IGF-I receptor immunoreactivity present in chloride cells
Gill
IGF-I mRNA levels increase after exogenous GH and transfer
to SW in salmonids and tilapia (O. mossambicus)
Exogenous IGF-I increases salinity tolerance, gill Na +,K +-ATPase
activity and development of chloride cells
IGF-I immunoreactivity present in chloride cells
McCormick (unpublished)
McCormick (unpublished)
Weng et al. (2000)
McCormick (1995)
(1998a)
Reinecke et al. (1997)
A large number of binding studies in fish have found evidence for a

single class of corticosteroid receptors (CR) (see references in Prunet et al.,
2006). However, in the last several years, molecular techniques have
demonstrated the presence of genes in several teleost species related to the
mammalian glucocorticoid (GR) and mineralcorticoid receptors (MR).
Fish GR has been characterized in several species (Oreochromis
mossambicus, Paralichthys olivaceus), with a second isoform present in some
species (O. mykiss, Haplochromis burtoni). In addition, MR has been
molecularly characterized in O. mykiss and H. burtoni. Using a transfected
cell line, Sturm et al. (2005) found that the rainbow trout MR (rtMR) has
high transactivation efficiency for both aldosterone and 11deoxycorticosterone (DOC), similar to the mammalian MR. Prunet et al.
(2006) suggest that DOC, present in the plasma of some teleosts at levels
that could activate the rtMR, might be a mineralocorticoid in fish. It may
be possible that the teleost MR is involved in the dual osmoregulatory
role (ion secretion and uptake) of cortisol in teleost fish. However, the
physiological function of the MR in fish and the possible physiological
relevance of DOC remains to be established.
506
Fish Osmoregulation
Table 16.5
Physiological evidence for a hyperosmoregulatory role of cortisol.
Action
References
Plasma
Transfer from SW to FW transiently increases plasma cortisol
levels

McCormick (2001)
Effects of cortisol treatment

Restored plasma osmolality and ion levels in hypophysectomized
eels, goldfish and bowfin
Increased surface area of gill chloride cells and the influx of
sodium and chloride in FW rainbow trout, tilapia, eel and catfish
Stimulated whole-body calcium uptake and the branchial calcium
pump in freshwater rainbow trout
Enhanced H+-ATPase activity in gills of salmonids, possibly
involved in sodium uptake in hypo-osmotic environments
Increased ion regulatory capacity after transfer of Sparus
aurata to low salinity environments
Stimulated gill Na+,K +-ATPase activity, plasma osmolality
and ion levels in BW-adapted S. aurata
Interactions with other hormones
A positive interaction of cortisol with PRL for maintenance
of ion balance in FW-acclimated channel catfish Ictalurus
punctatus and stinging catfish Heteropneustes fossilis
A positive interaction of cortisol with PRL for promoting the
transepithelial resistance and potential of cultured branchial
epithelia from FW rainbow trout
McCormick (2001)
Laurent and Perry (1990)
Perry et al. (1992)
Flik and Perry (1989)
Lin and Randall (1995)
Marshall (2002)
Parwez and Goswami (1985)

Eckert et al. (2001)
Zhou et al. (2003)
HORMONE INTERACTIONS
In addition to the independent osmoregulatory actions of PRL, GH/IGF-I
axis and cortisol, there is substantial evidence indicating the existence of
synergy and antagonism of these hormones with one another.
PRL and Cortisol
Consistent with its role in promoting acclimation to low environmental
salinities, PRL antagonizes the salt-secretory actions of both cortisol and
GH (O. mykiss: Madsen and Bern, 1992; S. salar: Boeuf et al., 1994; S.
trutta: Seidelin and Madsen, 1997). Seidelin and Madsen (1997) found
that PRL could reverse all of the increases in hypoosmoregulatory ability
induced by cortisol, but did not affect the capacity of cortisol to increase
gill Na+,K+-ATPase activity. They suggested that an interaction of PRL
and cortisol on salt secretory capacity may occur in non-branchial tissue
507
such as the intestine. Cortisol has been shown to rapidly decrease the
release of PRL from the tilapia pituitary (Borski et al., 1991).
As outlined above, cortisol also has an apparent role in ion uptake,
and there is evidence for a positive interaction of exogenous treatment
with cortisol and PRL for maintenance of ion balance in FW fish (Parwez
and Goswami, 1985; Eckert et al., 2001). In S. aurata, a greater activation
of pituitary PRL and ACTH cells have been shown to occur in BWacclimated fish relative to SW-acclimated fish, suggesting a possible
cooperation of PRL and cortisol in the control of osmoregulation at low
salinities (Mancera et al., 1993, 2002). Using an in vitro gill cell
preparation, it has been demonstrated that PRL and cortisol act
synergistically in order to promote transepithelial resistance and potential
(Zhou et al., 2003).
PRL and GH/IGF-I Axis
It has also been demonstrated that treatment with PRL can antagonize the
hyperosmoregulatory actions of GH and IGF-I in salmonids (O. mykiss:
Madsen and Bern, 1992; S. salar: Boeuf et al., 1994; S. trutta: Seidelin and
Madsen, 1997, 1999). This effect has been shown to occur at the level of
specific ion transporters (gill Na+,K+-ATPase), and chloride cell number
and morphology, resulting in differences in decreased whole animal
performance (higher plasma ions) in SW. This antagonistic action of PRL,
along with the lower PRL levels seen in BW, may explain the greater
efficacy of GH treatment on salt-secretory capacity in BW relative to FW
(Bolton et al., 1987; McCormick, 1996). Currently, it is thought that this
antagonism occurs primarily at target tissues, as we are not aware of any
studies indicating that GH and PRL affect one anothers synthesis or
secretion.
GH/IGF-I Axis and Cortisol
An important synergy of the GH axis and cortisol to improve salinity
tolerance and salt-secretory capacity has been demonstrated in salmonid
and non-salmonid species. This cooperation is mediated by increased
expression of gill Na+,K+-ATPase subunits, gill Na+,K+-ATPase activity,
and abundance of Na+-K+-2Cl cotransporter in gill chloride cells
(Madsen, 1990; McCormick, 1996; Mancera and McCormick, 1999; Pelis
and McCormick, 2001; McCormick, 2001). GH has been shown to
508
Fish Osmoregulation
increase the abundance of gill cortisol receptors in two species of

salmonids (O. kisutch and S. salar) (Shrimpton et al., 1995; Shrimpton and
McCormick, 1998), and this may explain a substantial part of the
interaction between GH and cortisol. Seidelin et al. (1999) found an
additive effect of IGF-I and cortisol on gill chloride cell number and
Na+,K+-ATPase activity, but to date, no one has examined whether IGF-I
can increase the number of gill cortisol receptors. Another possible
mechanism of IGF-I and cortisol interaction is through a possible antiapoptotic action of IGF-I on gill chloride cells, permitting cortisol to affect
a greater number of partially or fully differentiated chloride cells.
In addition to interactions at target tissues, GH, IGF-I and cortisol are
likely to interact so as to affect one anothers synthesis and secretion,
though surprisingly little research has been done in this area. GH has been
shown to increase the sensitivity of the interrenal tissue to
adrenocorticotropic hormone (ACTH) in vitro and in vivo, thus enhancing
cortisol release (Young, 1988). Exogenous cortisol has been shown to
decrease the circulating levels of IGF-I (Peterson and Small, 2005;
McCormick, unpublished results). It is important to remember that these
hormones have active roles in growth and energy mobilization, and thus
their feedback mechanisms may reflect their involvement in processes
other that just osmoregulation.
CONCLUSION
The control of the osmoregulatory system of teleosts involves several
hypophysial and extra hypophysial hormones (PRL, GH and cortisol),
which play an important role in osmotic acclimation (McCormick, 1995,
2001; McCormick and Sakamoto, 2006). It is a well-established fact that
PRL has an important role in the FW acclimation of many teleosts, though
the mechanisms of ion regulation controlled by this hormone have not
been fully elucidated. In contrast, the osmoregulatory role of the GH/IGF-I
axis appears to be more highly species-dependent. In salmonids this axis
has a hypoosmoregulatory role acting clearly as a SW-adapting hormone.
However, in non-salmonid species, the evidence is contradictory, with GH
exhibiting an apparent hypoosmoregulatory role in some species, and no
clear osmoregulatory role in others. Finally, cortisol has been shown to
have a role in SW acclimation in both primitive and advanced teleost fish.
However, in recent years, evidence also suggests a role for cortisol in ion
uptake in low-salinity water-adapted fish. This new evidence suggests a
509
dual osmoregulatory role for cortisol, with the classic role of stimulation
of ion secretion in hyperosmotic media (in cooperation with GH and
IGF-I), and an additional role of increasing ion uptake in hypoosmotic
environments (in cooperation with PRL).
Acknowledgements
The present publication was supported by a grant BFU2004-04439-C0201B (Ministerio de Educacin y Ciencia, Spain) to J.M.M.. The authors
would like to express their gratitude to the members of the laboratory of
Dr. Mancera: Dr. Ral Laiz-Carrin, Jose Mara Guzman, Francisco Jess
Arjona and Luis Vargas-Chacoff for their contribution to the
understanding of fish osmoregulation. We also thank Michelle Monette for
her comments and corrections on the manuscript.
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Fish Osmoregulation
Index
11b-hydroxysteroid dehydrogenase 207,

211
11-deoxycorticosterone 209, 211
1a-hydroxycorticosterone 122
3,5,3-triiodothyronine, T3 37, 41
6-n-propyl-2-thiouracil (PTU) 37
A
Acid excretion 192, 337, 368
Acidic water 70, 136-138, 143
Actin 167, 168, 181, 198, 214, 217, 250,
259, 347, 350, 377, 402, 404, 407, 413,
415-419, 471
Adrenocorticotropic hormone (ACTH)
46, 508
Aglomerular 88, 115, 159, 169, 251, 252,
267, 268, 312, 443
Albumin 37, 38
Aldosterone 10, 122, 207-209, 211
Alkaline lakes 136
waters 136
Amiloride 197, 362, 414, 415
Amino acid(s) 68, 91, 141, 282, 290, 418
metabolism 294, 297
Ammonia 241
excretion 183
aMSH 46
Angiotensin 85-87, 89-96, 98, 99, 101103, 106-108, 111-116, 119-122, 167,
168, 347
converting enzyme 89, 106

receptors 93, 94
Angiotensin I 87, 91
Angiotensin II 86, 90, 91, 102, 108, 167,
168
Angiotensin III 86
Angiotensinogen mRNA 86, 87, 89, 90,
92
Anion exchanger 36, 345, 362
Antarctica 250-252
Antibody 4, 6, 19, 205, 206, 342, 360,
363-367, 369-371, 373-379
Antidiuresis 114
Antigen-retrieval 382
Antisense 210, 212
Apical membrane 37, 196, 242, 341, 343,
345, 348, 369, 372, 396, 397, 405, 408,
410, 432, 442, 443
Apoptosis 12, 13, 15, 21, 189, 500
Aquaporin 202, 363, 372
Arginine vasotocin (AVT) 151, 153, 154,
158, 160, 162, 169, 170, 398, 399, 406
AT1 receptors 93, 96
Atlantic salmon 199, 215, 407, 502
ATP 282, 297, 298, 438, 442
ATPases 282, 431
Atrial natriuretic peptide 197, 399, 403,
405
518
Fish Osmoregulation
B
Basolateral membrane 36, 37, 162, 186,
195, 242, 396, 397, 405, 408, 409, 411,
412
Bicarbonate transporters 204
Binding sites 45, 46, 93-95, 107, 108, 112,
113, 116, 119, 120, 142, 167, 436, 502
proteins 37, 38, 40, 416, 431, 434,
460, 502, 505
Bioinformatics 199, 215
Blood pressure 86, 106-108, 111, 114, 115,
118, 121, 163, 165, 167, 314, 315
Brackish water 8, 72, 103, 111, 157, 259,
261, 262, 280, 395, 500
Bradykinin 87, 90, 107, 111
Brain 17, 42, 46, 48, 89, 90, 93-96, 107,
108, 154, 155, 209, 238, 254, 280, 403,
406
RAS 107, 108
Branchial Ca2+ uptake 72, 73, 430, 433,
436, 439, 457, 465, 466
Branchial tight junctions 142
Brush-border 194
C
Ca2+ exchange tissues 430
Ca2+ excretion 440, 441, 451, 452
Ca2+ homeostasis 429, 435, 445, 450, 453,
455, 457
Ca2+ in larvae 141-145, 437, 438, 446448, 470
Ca2+-ATPase 362, 396, 431, 434, 436,
438, 439, 442, 471
Ca2+-binding proteins (CaBPs) 434
Ca2+-sensing receptor (CaSR) 448
Cadmium 433, 435
Calcified tissue 443
Calcitonin 444, 451, 452, 457-459
Calcium (Ca2+) 72, 74, 88, 94, 112, 115,
183, 184, 197, 239, 242-245, 362, 372,
398, 401, 407, 409, 410, 412-414, 427,
470-472, 506
efflux 184
uptake 183, 184, 243, 244, 506
cAMP 166, 168, 321, 397-399, 402-405,
408-410, 412, 414, 416, 418
Capacity to export glucose 293, 294
Captopril 87, 90, 106, 107, 110, 111, 115,
116
Carbonic anhydrase 185, 343, 345, 363,
375, 381
Carbonic dioxide 240
Catecholamines 15, 50, 238, 240, 244,
297, 398
Cell culture 182, 183, 185-188, 212
isolation 189, 190, 192, 193
lines 179, 188, 189
subtypes 191-193
volume regulation 185, 189, 266,
411-413
Cellular dehydration 102, 109
differentiation 186
polarity 184
CFTR 348, 360, 365-367, 376, 379, 381
Chloride (Cl )45, 69, 70, 77, 78, 93, 94,
100, 101, 109, 110, 140, 141, 158, 160162, 164, 180-184, 187-190, 192-194,
204, 244, 245, 255, 259, 261, 349, 365,
367, 371, 372, 380, 383, 499-508
cells 69, 77, 93, 94, 140, 158, 160,
162, 180-184, 187-190, 192, 237,
259, 261, 349, 365, 367, 372,
380, 383, 499, 500, 503, 505-508
cotransporters 204
influx 184
secretion 183, 188
Cholesterol 195, 253
Chromaffin cells 50
Cichlid 208, 209
Circumneutral water 139
Circumventricular organs 107
Citraconic anhydride 383-385
Cl/HCO 3 exchange 342, 370
Index
Clearance 39, 503
Cleithrum 187
Cloning 185, 189, 190, 192, 193, 196, 376
Collecting duct 311, 368
Composition of fluids 337, 339
Composition of the fluid absorbed by the
intestine 346
Conjugated thyroid hormones 39, 191,
378, 380, 381, 383
Corpuscles of stannius 88, 243, 428, 449,
462, 464
Corticosteroid 121, 122, 206, 209-211,
502, 505
Corticotropin-releasing hormone (CRH)
17, 46, 47
Cortisol 10, 11, 14-17, 20, 21, 46-48, 50,
51, 120-122, 167, 181, 184-187, 191,
203, 207-211, 238, 239, 244, 263, 297,
347, 349, 350, 417, 418, 433, 452, 453,
455, 497, 498, 500, 502-509
CRH 17, 46, 47
Culture media 188
Cyclostomes 36, 37, 92, 96
Cystic fibrosis 204, 360, 365, 397
Cystic fibrosis transmembrane
conductance regulator 204, 397
Cystic Fibrosis Transmembrane Regulator
(CFTR) 360, 365
Cytokines 2, 4, 5, 13, 17, 22
Cytotoxicity 4, 5, 7, 10, 13, 15, 190
D
Deiodinases 40-43
Deiodination 39-43
Density gradient centrifugation 189, 190
Diet 1, 6, 8, 67-73, 75, 76, 141, 186
Dietary Ca2+ 72, 73, 75
Dietary Ca2+/phosphorus ratio 75
Dietary Mg2+ 76-78
Dietary Na+ 68, 69, 71
Dietary NaCl 69, 72
Dietary phosphorus 74, 75
519
Dietary salt 68, 69, 71, 72

Digestive tract 68, 186, 242
Dipsogenic responses 105
Distal tubule 164, 166, 311-313, 319, 322,
324-327
Dithiothreitol 42
Divalent ions 112, 286, 310, 311, 320,
321, 326, 327, 335, 338, 498
DNA-binding 208, 264
Drinking 68-70, 72, 101-111, 158, 167,
168, 186, 236, 334, 336-338, 340, 347,
348, 405, 498, 503, 504
rate 68-70, 72, 109-111, 236, 348
Dual role of the intestine: feeding vs
osmoregulation 346
E
Eel 67, 91, 94, 114, 116, 120, 190, 202,
258, 285, 348, 360, 361, 363, 373, 506
Eggs 144
Eicosanoids 188, 407
Elasmobranchs 90-92, 95, 96, 100-102,
108, 109, 111, 112, 117-119, 121, 123,
159, 267, 309, 310, 312-314, 316-319,
320, 322-324, 326, 327, 368
Electrolyte 158, 235, 236, 238, 240, 244,
279, 289, 290, 336, 343
Electrolytic disturbances 244
Endocrine control of Ca2+ balance 449,
451
Endocrine control of intestinal salt and
water transport 347
Energetic cost 278
Energy 237, 238, 257, 258, 261, 262, 277279, 281-287, 289-293, 295-299, 321,
323, 325, 501, 508
demand 279, 284-286, 289, 292,
295, 298, 299
metabolism 207, 218, 501
Enterocyte cell culture 186
Environmental salinity 48, 97, 98, 109,
155, 160, 181, 504
520
Fish Osmoregulation
Epithelial Ca2+ channel (ECaC) 362, 372,

433
Epithelial sodium 192, 196, 204, 362, 369
Epithelial Sodium Channel (ENaC) 192,
196, 204, 362, 369
Epithelioma Papulosum Cyprini (EPC) 188
Esophagus 334, 335
Estradiol 10, 16, 444, 452, 453, 455, 469
Estuarine 250, 252, 255, 260, 262-266,
269, 395, 410, 413
Euryhaline 71, 96, 100, 101, 103-106, 108,
109, 113, 123, 182, 259-261, 265, 266,
269, 279, 338, 341, 350, 396, 501
elasmobranchs 100, 109, 314,
317, 323
Euryhalinity 96
Excretion 39, 137, 139, 140, 237, 238,
240, 242, 244, 336, 499
Expressed sequence tag 201
Extracellular dehydration 102, 109
F
FAK 405, 411, 412
Feces 67, 68
Filtration rates 112, 113, 118, 237, 313
Fixation 376
Freshwater 237, 278, 338-341, 347-350
fishes 67, 74, 266, 368, 371
Fry 8, 106, 144
G
Gastrointestinal tract (GIT) 158, 164,
186, 289, 466, 500
Gene duplication 202, 203, 209
expression 89, 203, 210, 212, 414
fusion 208
silencing 212
Gill(s) 67, 89, 93-96, 102, 105, 158, 160163, 168, 169, 180, 182-184, 187-196,
203-206, 236, 244, 278, 282-285, 288,
295, 395-397, 401, 403, 404, 406-409,
411, 413, 414
cell culture 182

filament culture 184
Na+,K+-ATPase 93, 284
Na+,K+-ATPase activity 284
Gillichthys mirabilis 216, 396, 400
Gilthead seabream 279, 283, 287, 291,
292
Glomerular filtration 159, 165, 286, 313,
466
filtration rates 112, 466
receptors 93
Glomerulotubular balance 165
Glomerulus 87, 88, 93, 94
Glucocorticoid receptor 207, 211
Glucocorticoid responsive element 208,
211
Gluconeogenesis 287, 288, 290, 291, 293
Glucose 68, 280-291, 293, 294, 296-299,
345
capacity to export 293, 294
use 287, 291
oxidation 284
Glucose phosphorylating capacity 298
Glucuronidation 39, 40
Glycogen 285, 286, 288-290, 292, 293,
295-299
Glycogenolysis 160, 283, 287, 290-293,
295, 296
Glycogenolytic potential 285
Glycolytic 284, 286, 287, 293, 296, 298,
299
capacity 284, 286, 293
Goblet cells 186
Goitrogens 36
(G-protein)-coupled receptors 153
Gradient 138-140, 244
Granular epithelioid cells 88
Granulocytes 4
Growth 46, 47, 51, 68, 71-74, 76, 77, 120,
135, 142-146, 167, 183, 202, 210, 347,
350, 497, 498, 501, 502, 508
Index
hormone 46, 47, 51, 120, 167,
347, 350, 454, 497, 498, 501
Guanylins 347, 348
Gut 69, 105, 109, 111, 112, 160, 180, 186,
187
sacs 186
H
H+/K+-ATPase 362
Haematopoietic cells 51
Hard water 136, 141, 146
Hatching 142, 144, 145
Head-kidney 6, 46, 49-51
Heart 75, 89, 93-96, 154, 280, 299, 349,
403, 414
Heat-induced epitope retrieval (HIER)
382, 384
Heterotopic thyroid follicles 35, 49-51
Highly alkaline intestinal lumen 337
High-sodium diets 69
HOE-694 197
Hormone 35-49, 51, 88, 101, 120, 152,
153, 155-169, 181, 184, 186, 398
Hyper 43, 44, 100, 109, 263, 268
Hyperosmotic challenges 100
Hypersaline 6, 20, 21, 259-262, 266, 268,
280, 395, 428
water 280
Hyperthyroidism 37, 39, 42, 43, 47
Hypertonic 102, 277, 278, 400, 411, 412,
415, 419
Hypertonicity 189, 346, 405, 414, 419
Hypothalamic magnocellular 151
Hypothalamic neurosecretory neurons
152
Hypothalamus 35, 46, 47, 155, 169
Hypothalamus-pituitary gland-thyroid
axis 35
Hypothalamus-pituitary-interrenal axis
47
Hypothyroidism 37, 42, 43
521
Hypotonic 184, 185, 236, 333, 400, 401,

412, 415, 417-419
shock 184, 400, 402, 410-413,
415, 417-419
I
Ice goby (Leucopsarion petersii) 48
Immune system 1-5, 9, 10, 13-18, 20, 21
Immunoblotting 185, 364, 416
Immunocytochemistry 93, 94, 152, 360,
364, 372, 411
Immunology 190
Inner ring deiodination 42, 43
Insulin-like growth factor 350, 497, 498,
501
Interrenal cells 46, 50
gland 15, 95, 121, 122
Intertidal 250, 252, 262-266, 268, 269,
419
Intestinal anion exchange and Cl
absorption 342
Intestinal Ca2+ uptake 337, 437, 439, 461,
462, 469
Intestinal fluid composition 337, 338
Intestinal perfusion 186
Intestinal transport 333, 341, 345, 347,
348
Intestinal transport processesNaCl
absorption 341
Intestinal transport processeswater
absorption 345
Intestine 67-69, 72, 73, 78, 89, 93-96, 102,
111, 112, 121, 154, 158, 164, 186, 194,
237, 278-290, 333-341, 344-349, 414,
437-440, 445, 447, 449, 453, 457, 462,
466, 469, 471, 499, 503, 507
Intrarenal blood flow 115
Intrarenal RAS 115, 116, 118
Iodothyronine 37, 39, 40, 42, 43, 51
Iodothyronine deiodinases 41
Iodothyronine metabolites 39, 40
Ion channels 204, 253, 290, 396, 397, 410,
416, 431
522
Fish Osmoregulation
Ion transport 44, 71, 77, 78, 158, 183-185,

187, 189, 193, 194, 196, 198, 202, 204,
211, 277, 282, 318, 359, 360, 364-366,
372, 376, 395, 398, 406, 419
Ions loss 137
Ion-specific dyes 194
Isoform(s) 43, 120, 197, 202, 203, 205,
211
switching 202-204, 376
Isosmotic load 100
Isotocin 151-155, 157, 158, 160, 161, 168170, 407
J
JNK 405, 411, 412, 414, 416
K
Kidney 2, 3, 6, 7, 10, 15, 19, 21, 49, 50, 75,
76, 88-90, 93-96, 105, 114-118, 122,
123, 154, 158-160, 164-166, 168, 169,
180, 185, 187, 191, 193, 194, 236, 264,
268, 278, 279, 281, 286-290, 309-315,
317, 319, 322-327, 337, 360, 365, 368,
383, 406, 433, 440-443, 446, 449, 451453, 463, 468, 471, 498, 499, 503, 504
Na+,K+-ATPase activity 289
tubules 94, 160, 166, 317
Killifish 10, 38, 42, 103, 159, 162, 178,
183, 191, 205, 215, 217, 260, 262, 266,
321, 322, 350, 361, 368, 369, 372, 415418, 431, 434, 453, 464, 498, 501
Kinins 90
Knockout models 210
Kroghs principle 200
L
Lactate 254, 280-289, 296-299
oxidation 285, 289, 297
Lamprey(s) 36, 90-92, 96-101, 111, 118,
119, 123, 186, 309-320, 322, 324, 325,
327, 333, 373, 383
Larvae 141, 142-145, 367, 437, 438, 445448, 463, 469, 471
Laser scanning cytometry 191

Lead 14, 39, 76, 77, 257, 263, 286, 298,
322, 434, 435, 461
Lipogenesis 294
Lipolysis 294, 297
Lipolytic enzymes 299
Lipoproteins 38
Liver 280, 290
Local effector 209
Loop of Henle 237
Luminal alkalinity 337
Lymphocytes 2, 4, 5, 11, 13, 15, 21
M
Macrophages 2, 4, 13, 21, 379
Magnetic separation 191
Marine fish 6, 88, 100, 158, 161, 183, 186,
190, 266, 311, 313, 315, 318, 326, 333335, 337, 348, 435, 437, 466
Marker enzymes 196
Mass spectrometry 216
Medaka 92, 156, 199, 201, 202, 212, 218
Medulla oblongata 107, 108
Membrane lipids 195, 253
vesicle 185, 195, 196
Metabolism 36, 39, 40, 43, 75, 207, 218,
236, 240, 258, 277-279, 283, 285-287,
289-291, 294-299, 452, 454, 457, 459,
460, 462, 463, 465, 467, 501, 502
Metabolomics 217, 218
Metamorphosis 48
Methimazole (MMI) 37, 42
Microarray 44, 213, 214, 215
Migration 48, 96, 111, 119, 195, 444, 456
mineralocorticoid receptor 207
Missing cationacidic absorbate 346
Mitochondria-rich (MR) cells 367, 430
MLCK 405, 412, 414
MMI 37
Monovalent ions 237, 311, 335, 443, 498
Morpholino 212
Index
Mortality 8, 73, 77, 137-139, 142, 239,
240, 245
Mucus 3, 183, 186, 187, 219, 240, 244,
337, 381, 499, 501
Mudskippers 188, 263, 413
N
Na,K-ATPase 43, 44, 45, 51
Na+, K+-ATPase activity 284
Na,K-ATPase subunit 44
Na+,K+,2Cl symport 396
Na+,K+ -ATPase 36, 93, 117, 120, 121,
237, 284, 286, 289, 298, 299, 318, 334,
341, 360, 396, 405, 407, 409, 415, 499,
500, 502-508
Na+/Ca+ 242
Na+/Ca2+-exchanger 431, 432, 434, 435,
438, 439, 442
Na+/H+ exchange 185, 415
Na+/H+-exchanger (NHE) 361
Na+/I symporter (NIS) 36
Na+/K+-ATPase 77, 180, 186, 195, 198,
202-204, 360, 365-376, 380, 383-385,
431, 434, 439, 453
+
Na : HCO3 cotransporter (NBC) 362
Na+:K+:2Cl cotransporter (NKCC) 360,
365
Na+K+-ATPase and Na+:Cl Cotransporters 341
NaCl 68-72, 99, 144, 158, 164, 237, 240243, 251, 254, 256-259, 267, 310, 318326, 334, 336, 341, 343, 345, 401, 406,
410, 413, 414, 418, 419
Natriuretic peptides 10, 347, 349
Nephron 159, 166, 168, 169, 311, 312,
315, 318, 319, 322-324, 327
loops 312, 323
Neurohypophysial hormone 152-154,
156-164, 166-169
Neurohypophysis 152, 155, 169
Neurons 151, 152, 155, 169, 349, 401,
402, 404
Neuropeptide Y 348
523
Neuropeptides 151-153, 159

Neurophysin 152
Neutral waters 71, 136
NHE 361, 367, 368, 376
Nitric oxide 13, 407
NKCC 369, 371, 396, 502
Nonspecific cytotoxic cells (NCC) 4
Northern analysis 207
NPO (nucleus preopticus) 152
Nuclear magnetic resonance spectroscopy
218
Nucleus lateralis tuberalis 47
Nucleus preopticus, NPO 46
O
Ontogeny 48
Opercular epithelium 178, 417, 418
Opistonephros 49
Organic anion transporter 39
Osmoregulation 36, 44, 48-51, 213-215,
218, 219, 316, 498
Osmotic acclimation 201, 277, 279-283,
285, 287, 289, 290, 293-299, 501, 508
Osmotic water influx 102, 111, 112, 185
OSR1 405, 411, 412
Ouabain 36, 44, 45, 198, 434
Outer ring deiodination 40-43
Oxidation of glucose 284
Oxidation of lactate 285, 297
Oxygen 4, 21, 239, 240-242, 261-263,
265, 269, 278, 279, 283
consumption 261, 262, 278, 279
Oxytocin 152-154, 160, 161, 164, 168
P
Papaverine 87, 106, 109-111
Paracrine RAS 89, 90
Parathyroid hormone (PTH) 448, 451,
467
Paraventricular nucleus 47
Parr-smolt transformation 48, 49
524
Fish Osmoregulation
Pathogen 2-4, 13, 21

Pavement cell 44, 47, 48, 141, 183, 191193, 282, 396, 409, 411, 413
Peanut lectin agglutinin 191, 432
Pendrin 36, 362, 372
Pentose phosphate 283, 286, 287, 291,
294, 299
Pentose phosphate shunt 287
Perchlorate 36
Perfused kidney 90, 114
Peripheral thyroid hormone metabolism
40
Permeability 70, 77, 78, 195, 238, 453,
466, 498, 499
pH 71, 135-143, 146, 204, 241, 415
pH changes 136
Phagocytosis 4, 5, 7, 10-12, 15, 16, 19, 21
Pharmacology 190, 196, 205
Phenamil 197
Phospholipid microdomains 195
Pituitary pars distalis 46
PKC 411, 415, 417, 418
Plasma 69-74, 76-78, 86, 89, 90, 92, 97,
98, 100, 102, 103, 107-110
cortisol 121, 239, 349, 506
membrane calcium ATPase 372
osmolality 86, 97-100, 102, 103,
108, 109, 117, 157, 260, 262,
310, 338-340, 347, 350, 406,
413, 415, 500, 506
sodium 110, 242
thyroid hormone concentrations
36, 38
Polymerase chain reaction 213
Polyunsaturated fatty acids 253, 285
Potassium channel 214, 361
Preoptic area 46
Profit 245, 258
Prolactin 6, 46, 47, 167, 181, 198, 243,
347, 350, 417, 418, 452, 453, 497, 498,
500
Promoter region 43, 208
Pronephros 3, 15, 49
Protein 3, 4, 12, 36-38, 47, 87, 92, 93, 137,
153, 155, 161, 192, 195, 203, 205, 213217, 235, 237, 240, 252, 254, 257, 259,
263, 280, 282, 285, 295, 299, 344, 350,
360-365, 367, 371, 372, 376-378, 381383, 397, 404, 405, 408-412, 416, 418,
419, 427, 428, 433, 443, 444, 453, 455457, 463, 464, 467, 468, 471
Proteomics 215, 217
Proton pump 192, 196, 204-206, 368
Proximal tubule 90, 94, 159, 185, 311,
312, 317-322, 325, 327, 368, 466, 471
PTH-like peptides 467, 471
PTH/PTHrP 452, 467, 472
PTU 37, 41, 42
Pufferfish 87, 88, 199, 200, 202, 206, 433,
449, 467
Pyloric caecae 186
Pyloric ceca 68, 69
R
Radioimmunoassay 92, 467
Radiotracers 194
Rainbow trout 6, 8-10, 13, 15, 20, 38, 4244, 46, 48, 67-71, 73, 75, 77, 86, 87, 91,
93, 94, 96, 107, 108, 113, 116, 137,
138, 140-144, 155-157, 160-164, 183,
184, 187, 188, 190-192, 199, 202-205,
207-209, 212, 213, 215, 216, 253, 268,
280, 283-285, 287-297, 315, 316, 326,
342, 401, 404, 406, 407, 411, 432-434,
436, 441, 449, 453-459, 461, 465, 501,
505, 506
Reabsorption 76, 78, 112, 115-117, 166,
237, 251, 316-320, 322-327, 433, 441443, 451, 454, 462, 466, 471, 499
Rectal gland 93, 95, 96, 102, 117, 119,
122, 178, 195, 365
Red muscle 295
Renal Ca2+ uptake 442
Renal cell culture 185
Renal function 102, 112, 114, 116, 118,
119, 159, 164, 185, 186, 252, 335, 466
Index
Renal proximal tubule 90, 185
Renal tubular Na+, K+-ATPase 117
Renin 85-88, 90, 92, 96, 102, 115, 167
Renin-angiotensin system 85, 90, 96, 102,
167
Reporter gene 207, 208
Respiratory alkalosis 140
Respiratory chain 295
Ribozymes 212
S
Saline tolerance 103
Salinity 7, 97, 98, 100, 103, 105, 109, 155157, 160, 162, 169, 181, 191, 195, 198,
212, 236, 260, 277-282, 284-286, 289,
292, 293, 296, 298, 299, 314, 324, 338340, 406, 417-419, 499, 504-506
Salmonids 6, 18, 19, 21, 38, 44, 46, 48, 76,
368, 369, 444, 456, 459, 464, 501, 503508
Salt 68-72, 85, 97, 120, 122, 158, 160,
164, 168, 177, 179, 187, 195, 199-201,
206, 213, 215, 235, 237, 239, 240, 242,
245, 256, 259-262, 264, 266-268, 309,
310, 317, 324, 333-335, 347-350, 401,
405, 409, 411, 454, 502, 506, 507
Sauvagine 47, 403
Scyliorhinus canicula 95, 105, 365
Sea bass 161, 183, 257, 259, 280, 281, 284,
285, 296, 406, 407
Seawater 6-8, 40, 44, 72, 73, 75, 76, 104,
105, 123, 140, 157, 158, 160-162, 164,
168, 177, 181, 183, 188, 191, 192, 195,
201, 203, 214, 215, 237, 238, 250, 251,
253-255, 259-261, 264-269, 278, 279,
295, 296, 309, 310, 314-318, 322, 325327, 336, 339, 364, 365, 367, 368, 372,
396, 403, 410, 428, 437, 439, 463, 503
acclimation 44, 158, 181, 192,
214, 350
fishes 254, 326
Secretion 38, 45, 46, 89, 112, 114, 119,
121, 152, 155-159, 161, 162, 167, 169,
183, 185, 187, 188, 193, 251, 256, 259,
525
261, 264, 266, 311, 317, 319-322, 326,

327, 335, 337, 341, 342, 344-348, 365,
395, 397-404, 406-411, 413-416, 418,
419, 440-443, 448-450, 452-454, 458,
459, 464, 465, 468, 471, 499, 502-505,
507-509
Sequencing 86, 88, 199-201, 219
Skin 3, 67, 94, 160, 187, 188, 256, 396,
400, 402, 403, 450, 471, 499
cell culture 187, 188
Small interfering RNA 212
Sockeye salmon 186, 203, 459
Sodium 44, 69, 184, 190, 197, 198, 203206, 239-245, 255, 282, 290, 362, 367369, 383-385, 399, 434, 500, 506
channel 69, 192, 196, 204, 206,
368, 369, 500
transport 184, 189, 190, 192, 193,
196, 204, 206
Sodium pump (Na+/K+-ATPase) 44, 180
Sodium/calcium exchanger 197
Soft water 137-139, 432, 433, 436
Somatolactin 243, 349, 454, 455
Somatostatin 119, 349, 398, 399, 403, 418
SPAK 405, 411, 412
Spawning 49, 137, 295, 444, 456, 459
Splice variants 208
Stanniocalcin 243, 449, 452, 462, 464,
472
Steroid receptors 206
Steroid-binding assays 207, 209
Stickleback 92, 103, 155, 199
Stop-flow fluorimetry 194
Stress 13, 71, 157, 201, 238-241, 243, 261,
263, 264, 266, 269, 279, 281, 295, 297299, 334, 405, 410, 411, 413, 454, 455,
502
axis 35
response 46, 238, 263, 269, 405,
411, 455
Stressor 238, 239, 244, 265, 299
Sulfate excretion 185
Sulfation 39, 40
526
Fish Osmoregulation
Sulfotransferases 39
Suppressive subtractive hybridization 214
Surface enhanced laser desorption/
ionization 216
Survival 9, 15, 72, 135-137, 140-146, 201,
239, 445, 447, 449, 498
T
T3 37-45, 47-49, 51
Taurine 141, 185, 189, 282
Tegument permeability 240
Teleosts 13, 36, 37, 39, 44, 46, 49, 88, 90,
91, 94, 96, 100, 102-108, 111-118, 120,
121, 123, 135, 137, 152, 155, 158, 164,
165, 167, 182, 185, 195, 202, 243, 250,
256, 258, 259, 265, 267-269, 309-322,
324-327, 333, 335, 338, 341, 342, 344,
346, 348, 349, 395, 400, 401, 403, 404,
410, 414, 417-419, 428, 499
Thiocyanate 36
Thiourea 48, 49
Thyrocyte 36, 37
Thyroglobulin 36, 37
Thyroid gland 35, 36, 38, 45, 47-49, 457
Thyroid hormone(s) 10, 35-39, 43-45, 4749, 51
Thyroid hormone receptor 43, 48
Thyroid peroxidase (TPO) 37
Thyroid response element 43
Thyroidectomy 47
Thyroid-stimulating hormone 45
Thyrostatic thiourea 48, 49
Thyrotropin-releasing hormone (TRH)
45
Thyroxine 37, 50
Thyroxine-binding globuline (TBG) 37
Tilapia 36, 39, 42, 44, 46, 47, 51, 72-74,
77, 103, 137, 140, 141, 145, 180, 205,
214, 260, 280-283, 285, 290-294, 296,
298, 299, 338-340, 348, 360, 363, 367369, 371, 373, 383, 396, 400, 403, 404,
417, 437, 449, 453-456, 460-463, 465,
469, 470, 500, 501, 504-507
Tissue explant 186

Toadfish salinity tolerance 340
Transcription factors 214
Transcriptomics 213, 215, 217
Transfection assay 207
Transgenics 210
Transmission electron microscopy 192
Transport 93, 112, 115, 117, 120, 179,
182-185, 187-190, 192-198, 202, 204206, 211, 216, 237, 239-245, 253, 262,
277, 282, 333-336, 341, 343-350, 359,
360, 366, 395, 396, 398, 401, 405, 406,
408-412, 414, 415, 417-419, 430-432,
434-436, 438, 440-443, 445, 446, 449451, 453-455, 458, 462, 465-467, 469472
Transport processes in the intestinal
epithelium 343
Transportation 235, 239, 240, 242, 244,
245
Transthyretin (TTR) 37
TRH 46
Tricarboxylic acid cycle 295
Triglyceride 280, 290
Trunk-kidney 49, 50
TSH 45-47, 50
TSH b-subunit 45, 46
TTR 37, 38
Two-dimensional polyacrylamide gel
electrophoresis 215
U
UDP-glucuronyltransferases 39
Ultimobranchial glands 457, 459
Unstable habitats 249, 262
Unstirred water layer 193-195, 219
Urea 100, 101, 109, 110, 117, 122, 140,
141, 156, 159, 164, 168, 169, 195, 267,
310, 312, 323, 324, 327, 363, 372
transporter (UT) 169, 324, 363,
372
Urinary bladder 94, 166, 168, 178, 310,
311, 319, 325-327, 443, 449
Index
Urine 39, 67, 68, 70, 90, 112, 114-119,
137, 158, 165, 166, 169, 185, 236, 237,
286, 288, 310, 335, 336, 440-443, 497,
499
Urotensin 47, 349, 350, 398, 399, 403, 419
Urotensin I 47, 399, 403, 419
Urotensin II 349, 350, 398, 399, 403
Ussing chambers 185, 349, 439, 469
V
V1-type receptors 160, 161, 162
V2-type receptors 160
Vacuolar type proton ATPase 368
Vascular perfusion 119, 195
Vasoactive intestinal polypeptide (VIP)
348, 398, 404
Vasopressin 152-155, 157, 160, 167-169
V-ATPase 361, 368-370, 372, 376, 379,
380, 382-385
Vitamin D3 451, 452, 459-462
Vitellogenin 38, 428, 456
Volume depletion 97, 106, 123
Volume expansion 97
527
Volume receptors 97
W
Water hardness 136, 140-142, 144-146
influx 2, 102, 111, 112, 185, 240,
242, 244, 314
quality 146, 240
White muscle 295-297
Whole animal balance of water 336
Winter flounder 105, 106, 159, 185, 196,
320, 345, 348, 440-442, 449, 453, 462,
466, 471
Y
Yolk-balls 180-182
Yolk-sac 180, 181
Z
Zebrafish 36, 48, 87, 88, 92, 199, 200, 202,
205, 210, 212, 369, 404, 433, 447, 448,
467
Zinc 208, 433, 435
Zinc fingers 207, 208

Fish Osmorregulation

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Fish Osmorregulation

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Fish Osmoregulation

Universidade Federal de Santa Maria

Juan Miguel Mancera

Formerly Professor of Zoology

CIP data will be provided on request.

Fish lives in environments with a wide variety of chemical characteristics

5. Effect of Water pH and Hardness on Survival

6. Arginine Vasotocin and Isotocin: Towards

7. Cellular and Molecular Approaches to the

9. Special Challenges to Teleost Fish Osmoregulation

12. Intestinal Transport Processes in Marine

13. The Use of Immunochemistry in the Study of

14. Rapid Regulation of Ion Transport in

15. Control of Calcium Balance in Fish

16. Role of Prolactin, Growth Hormone, Insulin-like

Alberto Cuesta et al.

have on the fish immune responses, following by the importance and

Alberto Cuesta et al.

Maule and Schreck (1987)

Chou et al. (1999)

Domnguez et al. (2004)

Plante et al. (2002)

Table 1.1 Effect of salinity disturbances on fish immune responses.

Alberto Cuesta et al.

Alberto Cuesta et al.

pathogenic potential or survival of pathogens and not with the fish

namely PRL, GH and cortisol, have been shown to act as fish

Alberto Cuesta et al.

Sakai et al. (1996b)

Narnaware et al. (1998)

Sakai et al. (1996c)

Yada et al. (2001, 2004b)

Nevid and Meier (1995)

Yada et al. (1999)

Cuesta et al. (2005b)

Calduch-Giner et al. (1995)

Sakai et al. (1995, 1996c, 1997)

Sakai et al. (1996b)

Kajita et al. (1992)

Yada et al. (1999)

Yada et al. (2004b)

Sakai et al. (1996a)

Sakai et al. (1997)

Sakai et al. (1995, 1996c)

Cuesta et al. (2005b)

(Table 1.2 contd.)

(Table 1.2 contd.)

Tripp et al. (1987)

Law et al. (2001)

Wang and Belosevic (1995)

Esteban et al. (2004)

Watanuki et al. (2002)

Zou et al. (2000)

Saeij et al. (2003)

Cuesta et al. (2005b)

Weyts et al. (1997, 1998a)

White and Fletcher (1985)

Nevid and Meier (1995)

Alberto Cuesta et al.

(Table 1.2 contd.)